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https://f1000research.com/articles/9-1015/v1	20 Aug 20	{ "type": "Data Note", "title": "The complete genome sequence of Toxicodendron radicans, Eastern Poison Ivy", "authors": [ "Toby Pirro", "Stacy Pirro", "Toby Pirro" ], "abstract": "Eastern Poison Ivy (Toxicodendron radicans, Anacardiaceae) is well known in Eastern North America for causing contact dermatitis, an itchy and painful rash in most people who come in contact with it. We present the whole genome sequence and annotation of this species. A total of 96,255,779 paired-ends reads consisting of 28.9 G bases were obtained by sequencing one leaf from a wild-collected plant. The reads were assembled by a de novo method followed by alignment to related species. Annotation was performed via GenMark-ES. The raw and assembled data is publicly available via GenBank: Sequence Read Archive (SRR10325927) and Assembly (GCA_009867345).", "keywords": [ "Toxicodendron radicans", "Eastern Poison Ivy", "genome", "assembly", "annotation" ], "content": "Introduction\n\nEastern Poison Ivy (Toxicodendron radicans, Anacardiaceae) is well known in the Eastern United States, Canada, Mexico and parts of China for causing contact dermatitis, an itchy and painful rash in most of the human population (Barceloux, 2008). The rash, along with accompanying blisters, is caused by urushiol, an oil compound in the plant's sap. Urushiol-induced allergic rashes are a Type IV hypersensitivity reaction (Kalish & Johnson, 1990). This type of reaction is a cell-mediated response and can take hours to days to produce symptoms (Williams et al., 1999). Approximately 15% of people have no allergic reaction to urushiol, but most people experience a reaction from between 5–12 days after exposure. Allergic reactions can increase in duration and severity after each incident (Bonnekoh et al., 2019).\n\nExposure to the urushiol in Toxicodendron radicans can cause hypersensitivity in related plants, such as mango, Mangifera indica (Yoo & Carius, 2019), and the Chinese Lacquer Tree, Toxicodendron vernicifluum. Consumer products made from the Chinese Lacquer Tree are manufactured by curing the urushiol-containing sap to a clear, hard, waterproof substance. Improperly cured products can cause contact dermatitis in urushiol-hypersensitive people several years after manufacture, and present an ongoing health challenge to lacquerware workers (Ma et al., 2012).\n\nA complete genome sequence for this species will allow the insight into the evolution of the urushiol biosynthetic pathway.\n\n\nMethods\n\nA leaf from a single wild-collected Toxicodendron radicans plant was used as the source of genomic DNA. Extraction was performed on tissue from a single leaf using the Qiagen DNAeasy genomic extraction kit for plants, using the standard process. A paired-end sequencing library was constructed using the Illumina TruSeq kit, according to the manufacturer’s instructions. The library was sequenced on an Illumina Hi-Seq platform in paired-end, 2 × 150bp format.\n\nThe resulting fastq files were trimmed of adapter/primer sequence and low-quality regions with Trimmomatic (v0.33) (Bolger et al., 2014). The trimmed sequence was assembled by SPAdes (v2.5) (Bankevich et al., 2012) followed by a finishing step using RagTag (v1.0.0) (Alonge, 2020) to make additional contig joins based on conserved regions in related plant species: Mangifera indica (mango, GCA_011075055) and Pistacia vera (pistachio, GCA_008641045). Default procedures were used for all assembly steps.\n\nAnnotation was performed using GeneMark-ES (v2.0) (Lomsadze et al., 2005). Annotation was performed fully de novo without a curated training set and default parameters.\n\n\nResults\n\nThe genome assembly yielded a total sequence length of 454,874,194 bp over 270,263 scaffolds with an N50 of 1,945,245. The GeneMark-ES annotation resulted in 42,021 genes.\n\n\nData availability\n\nRaw and assembled data is publicly available via GenBank:\n\nRaw genome of Toxicodendron radican, Accession number SRR10325927: https://www.ncbi.nlm.nih.gov/sra/?term=SRR10325927\n\nAssembly of Toxicodendron radican, Accession number GCA_009867345: https://www.ncbi.nlm.nih.gov/assembly/GCA_009867345.1/", "appendix": "References\n\nAlonge M: Ragtag: Reference-guided genome assembly correction and scaffolding. GitHub archive. 2020. Publisher Full Text\n\nBankevich A, Nurk S, Antipov D, et al.: SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing. J Comput Biol. 2012; 19(5): 455–477. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarceloux DG: Medical Toxicology of Natural Substances: Foods, Fungi, Medicinal Herbs, Plants, and Venomous Animals. John Wiley and Sons. 2008. Publisher Full Text\n\nBolger AM, Lohse M, Usadel B: Trimmomatic: A flexible trimmer for Illumina Sequence Data. Bioinformatics. 2014; 30(15): 2114–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBonnekoh H, Poch G, Metz M: Severe contact dermatitis caused by urushiol in Japanese lacquer. Contact Dermatitis. 2019; 80(1): 55–56. PubMed Abstract \| Publisher Full Text\n\nKalish RS, Johnson KL: Enrichment and function of urushiol (poison ivy)-specific T lymphocytes in lesions of allergic contact dermatitis to urushiol. J Immunol. 1990; 145(11): 3706–3713. PubMed Abstract\n\nLomsadze A, Ter-Hovhannisyan V, Chernoff YO, et al.: Gene identification in novel eukaryotic genomes by self-training algorithm. Nucleic Acids Res. 2005; 33(20): 6494–6506. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMa XM, Lu R, Miyakoshi T: Recent advances in research on lacquer allergy. Allergol Int. 2012; 61(1): 45–50. PubMed Abstract \| Publisher Full Text\n\nWilliams JV, Light J, Marks Jr JG: Individual variations in allergic contact dermatitis from urushiol. Arch Dermatol. 1999; 135(8): 1002–1003. PubMed Abstract \| Publisher Full Text\n\nYoo MJ, Carius BM: Mango Dermatitis After Urushiol Sensitization. Clin Pract Cases Emerg Med. 2019; 3(4): 361–363. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "73876", "date": "13 Nov 2020", "name": "Rongxi Sun", "expertise": [ "Reviewer Expertise Genetic diversity and phylogeography" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 now thoroughly reviewed the manuscript “The complete genome sequence of Toxicodendron radicans, Eastern Poison Ivy”. Given urushiol in this plant’ sap causing dermatitis, an itchy and painful rash in most of the human population, the new assembly of complete genome sequence of this species helps to understand the evolution of the urushiol biosynthetic pathway. However, there are still some flaws in the manuscripts which the authors may consider to change:\nMethod: What is the source of the leaf used for sequencing, such as tree age and specific location.\n\nResults：How accurate is this high-throughput genome sequence? Whether there are indicators related to accuracy in the results.\n\nIn results, the annotation should be more discussed.\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? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "87604", "date": "24 Jun 2021", "name": "Xuming Zhou", "expertise": [ "Reviewer Expertise Genomics", "Phylogenetics", "Aging" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 reported the complete genome sequence of Toxicodendron radicans, which could help future research of this species. The manuscript is well written and should be considered for indexing prior to two questions:\nThe author should provide more details about the annotation, for example, how de novo and homologous approaches were performed.\n\nThe author should compare their genome quality to their relative species.\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? No\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "87603", "date": "07 Jul 2021", "name": "Hyungtaek Jung", "expertise": [ "Reviewer Expertise Genomics and Bioinformatics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have assembled the genome by employing Illumina sequencing technology using the leaf tissue of T. radicans. There is no doubt that the data generated in the current study will make a valuable contribution to the scientific community, however, I have several concerns about the analyses and/or the interpretation of the data as it currently stands.\nMethods: The fact is Illumina-based genome assemblies contain many misassemblies. Given the circumstances, why did authors choose Illumina reads over PacBio and Oxford Nanopore? What’s the overall sequencing and assembly summary? Any chloroplast DNA contamination? If yes, how did you treat them? Where are the executed scripts? Annotation with GeneMark-ES without any RNA-Seqs?\nResults: How did you evaluate the assembly accuracy (e.g. BUSCO)? Where are the structural (RepeatMasker) and functional annotations? It needs more explanation.\n\nIs the rationale for creating the dataset(s) clearly described? Partly\n\nAre the protocols appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-1015
https://f1000research.com/articles/9-1010/v1	20 Aug 20	{ "type": "Research Article", "title": "Associations between genetic factors in individualization of visual perception and components of event-related potentials during assessment of emotional visual stimuli (scenes) with distinct second-order features", "authors": [ "Pavel N. Ermakov", "Elena V. Vorobyeva", "Ekaterina M. Kovsh", "Alexander S. Stoletniy", "Magomed M. Dalgatov", "Fatimet P. Khakunova", "Asya K. Bersirova", "Pavel N. Ermakov", "Elena V. Vorobyeva", "Ekaterina M. Kovsh", "Magomed M. Dalgatov", "Fatimet P. Khakunova", "Asya K. Bersirova" ], "abstract": "Background: The aim of this paper is to investigate the associations between polymorphisms in the BDNF, COMT, and HTR2A genes with peculiarity of visual perception. In particular, how the carriers of different genotypes of Indicated genes emotionally evaluating visual scenes with distinct second-order features (images modulated by contrast) and how corresponding process is reflected in event-related brain activity (ERP). Methods: The study involved students who underwent PCR-based genetic analysis with the release of BDNF, COMT, and HTR2A genotypes. Participants were asked to emotionally assesse the specific stimuli – visual scenes that were generated from contrast modulations. At the same time the EEG were recorded using a 128-electrodes system. The average frequency of responses and ERPs for different emotional evaluations (negative, neutral and positive) were analyzed. Results: The study showed the BDNF Val/Val polymorphism was associated with the increase in the P2 amplitude in the occipital regions compared to the Val/Met genotype regardless of emotional evaluation. The COMT Met/Met genotype polymorphism associated with the increase of N170 negativity in the occipital regions during evaluation task. The HTR2A polymorphism A/A associated with increase in the P1 amplitude when positive or negative assessment were chosen, and decrease of later positive peak when neutral evaluation was chosen. Conclusions: The results suggested that emotional evaluation and recognition of visual scenes with distinct second-order features, as well as various strategies for processing visual information, reflected in amplitude and latency of different ERPs components and associated with the different genotypes of BDNF, COMT, and HTR2A genes. The indicated interconnections can act as genetic basis of individualize the mechanisms of visual perception.", "keywords": [ "Second-order visual features", "BDNF gene", "COMT gene", "HTR2A gene", "visual scenes", "visual perception", "emotions", "event-related potentials", "emotional assessment" ], "content": "Introduction\n\nDespite the fact that at this stage of evolution, the human visual system is the main channel for receiving information that performs complex processes of encoding and decoding stimuli, the mechanisms for recognizing and categorizing visual images still remain under-investigated (Babenko et al., 2011; Graham, 2011; Wilson & Gelb, 1984). In particular, second-order visual features – non-local characteristics of the perceived image, which including various options for contrast, orientation, and spatial frequency – are of special interest to human’s visual system researchers (Babenko et al., 2011; Babenko et al., 2016) These features are image regions characterized by spatial heterogeneities of brightness gradients – the first-order characteristics. These heterogeneities are distinguished by the so-called second-order filters, which detect regions with spatial modulations of contrast, orientation, and spatial frequency in the images. First-order visual mechanisms form the basis for subsequent spatial grouping. This is a multi-channel system that performs local analysis of the orientation of the brightness gradients at various spatial frequencies. Second-order visual mechanisms detect spatial changes and represent a certain set of filters.\n\nA number of experimental studies demonstrated that second-order visual mechanisms are selective towards modulations of contrast, orientation (surface curvature) and spatial frequency (depth) and have different cortical localization. The search for second-order stimuli is pre-attentive (Yavna & Babenko, 2012; Yavna et al., 2012).\n\nSecond-order features are known to play an important role in identifying images. The studies aimed at studying the role of second-order visual mechanisms in the recognition of emotional stimuli (especially scenes) are of particular importance today. Scenes are complex visual stimuli. Compared to the mechanisms of visual perception of faces and objects, little attention has been devoted to studying the brain mechanisms underlying processing of scenes (Aftanas et al., 2001; Codispoti et al., 2007; Delplanque et al., 2004; Mavratzakis et al., 2016; Olofsson et al., 2008; Pegna et al., 2004). Due to the fact that the human visual system often recognizes the scenes as combinations of images that include various elements, the study of these mechanisms can help us to clarify our understanding of the process of visual perception in vivo.\n\nAt the same time, we should take into account the influence of individual characteristics on the processes of visual perception. These may include psychological and biological characteristics. The genetic factors in the individualization of visual perception, i.e. genes that can affect the mechanisms of visual perception of images (including emotional scenes) are the catechol-o-methyltransferase (COMT) gene, the brain derived neurotrophic factor (BDNF) gene, the serotonin receptor gene (HTR2A), etc. In the science periodicals numerous studies examine the associations between these genes and individual psychological characteristics, including emotional intelligence, aggression, anxiety, and neuroplasticity and describe neurochemical processes characteristic to carriers of various genotypes. However, the influence of genes on the processes of visual perception, including the mechanisms for recognizing and evaluating (appraising) emotional visual stimuli (scenes) with the distinct second-order features are poorly described. This fact determined the topic of the present investigation. Let us now consider in more detail the functions of these genes.\n\n\nBDNF - the brain derived neurotrophic factor gene\n\nBDNF is a key molecule involved in the plastic changes associated with learning and memory. The BDNF stimulates the growth of neurons, axons, and dendrites, the formation of synapses and other processes of neuroplasticity not only in early ontogenesis, but also in the brain of the adult organism (Antal et al., 2010). Changes in BDNF expression in the brain structures involved in memory functions and perception processes (Miranda et al., 2019) are associated with human normal and pathological aging, as well as with mental illness. Together with other neurotrophic factors, BDNF plays an important role in the processes of adaptation of brain neurons to various external impacts influences, as well as in their growth, differentiation, and survival (Popova et al., 2017).\n\nIt is established that visual experience in the early postnatal period affects the maturation of the visual cortex. Furthermore, it was investigated the characteristics of maturation and plasticity of the visual cortex in mice with elevated level of BDNF (Huang et al., 1999). Under experimental conditions of visual deprivation, the authors found that BDNF overexpression is sufficient for the development of normal activity of the visual cortex in the absence of visual experience. Meanwhile, a decrease in BDNF expression slows down the development of visual areas of the cerebral cortex under conditions of visual deprivation (Gianfranceschi et al., 2003).\n\nCarriers of the BDNF Val/Val genotype with higher cortical plasticity were characterized by a more thorough processing of visual images. This may be explained by a more detailed analysis of the components of visual images which is associated with a higher activation of occipital areas. There is also some evidence that carriers of BDNF Val/Val genotype with higher neural plasticity perform visual-motor adaptation tasks worse than carriers of Val/Met genotype (Ermakov et al., 2017).\n\nCompared to carriers of the Val/Val genotype, those with the Met allele of the BDNF gene demonstrate higher activation in amygdala and hippocampus when recognizing individuals expressing emotions (Vorobyeva et al., 2019). These data indicate the contribution of the BDNF gene to the formation of brain mechanisms underlying anxiety, as well as its possible role in the recognition of emotional stimuli.\n\nAndreatta et al. (2019) showed that carriers of the Met allele of the BDNF gene have a higher level of anxiety. Loewenstern et al., (2019) confirmed this result, providing the following physiological data: the highly anxious carriers of the Met allele of the BDNF gene have stronger functional associations between the left amygdala and the right dorsolateral prefrontal cortex (anxiety phenotype).\n\nAccording to (Schofield et al., 2009), carriers of the Met/Met genotype of the BDNF gene had higher verbal recall errors and showed increased P300 latency when performing tasks in the oddball paradigm with corresponding alterations in hippocampal and lateral prefrontal activation, and a localized reduction in hippocampal gray matter. Thus, the Met allele affects the fronto-hippocampal systems involved in the selective processing of stimulus information and memory renewal in healthy individuals. Gajewski et al., (2012) described the impact of the Met/Met genotype of the BDNF gene on fronto-striatal associations. Meanwhile, this genotype was associated with a decrease in the severity of the Stroop effect in elderly people, which was accompanied by an increase in the amplitude of the N450 component during task performance. Researchers concluded that carriers of the Met/Met genotype of the BDNF gene were more resistant to interference. In their earlier work (Gajewski et al., 2010) also suggested that superior memory-based task-switching performance in elderly Met-allele carriers manifested itself in that N2 was increased while the P3 was decreased in Met-allele carriers. Beste et al., (2010) associated the Met allele of the BDNF gene with fewer errors during task performance, as well as with a less time stability of information stored in iconic memory (Beste et al., 2011).\n\n\nCOMT - the catechol-o-methyltransferase gene\n\nThis gene is associated with the dopamine system of the human brain. The enzyme catechol-O-methyltransferase regulates the transmission of impulses in the nervous system, and therefore can affect various mental processes (Serrano et al., 2019). Carriers of the COMT Met allele differentiate emotions and understand facial expression significantly better than those with the Val allele (Gohier et al., 2014b; Weiss et al., 2014). The structural features of this gene are associated with the activity of working memory (Barnett et al., 2011; Dumontheil et al., 2011; Gosso et al., 2008), multitasking ability (Rosa et al., 2010), anxiety (Hashimoto et al., 2007), extraversion (Reuter & Hennig, 2005) level of organization of regulatory (management) functions (Barnes et al., 2011), impulsivity and novelty seeking (Golimbet et al., 2006), and also the tendency to use psychoactive substances (Lovallo et al., 2019).\n\nIndividuals with a high enzymatic activity of COMT (the Val/Val genotype of the COMT gene) are better able to deal with stressful situations, because excess dopamine activity is quickly neutralized. On the other hand, individuals with a low enzymatic activity of COMT (the Met/Met genotype of the COMT gene) are more capable to develop complex plans, calculate the probabilities of events and think about their consequences; they have better memory, the ability to synthesize new information, but in a calm environment, in the absence of stress (Stein et al., 2006).\n\nThe data on the associations between the COMT gene genotypes and the brain correlates for facial expression recognition indicate that male carriers of the Met allele have higher values of latency of evoked potentials during perceiving faces; women with this genotype have a more pronounced arousal effect (Tamm et al., 2016). Bender et al., (2012) found that the DAT1 and COMT genes affect the same specific time interval (early contingent negative variation, CNV) associated with preparing for movement. According to (Gómez et al., 2003), the generators of this component are located in the frontal cortex (anterior cingulate gyrus) and in the occipital-temporal visual cortex, which corresponds to the expression of the COMT gene. There is evidence for the linkage of the region of chromosome 22 containing the COMT gene with an integrated phenotype that includes visual-motor dysfunction (Alfimova & Golimbet, 2011).\n\nKondo et al. (2012) discovered the effect of the COMT gene on speech perception reflected in the induced brain activity, as well as the impact of HTR2A gene on the perception of inverted figures. Later, these authors examined associations between COMT genotypes and recognition accuracy, as well as associations between HTR2A genotypes and the response time for recognition (Kondo et al., 2015).\n\n\nHTR2A - the serotonin receptor gene\n\nHTR2A has an inhibitory effect in the visual cortex. It is believed to be associated with episodic memory (Reynolds et al., 2006) and emotional intelligence (Kosonogov et al., 2019). Greater activation of the parieto-occipital areas of the brain during positive or negative judgment of stimuli by carriers of the dominant homozygous G/G genotype of the HTR2A gene may be explained theirs detailed assessment of stimulus images. Along with his, carriers of the recessive homozygous A/A genotype had similar activity in the same areas during positive emotional assessment of stimuli, which can be interpreted same way as G/G genotype (Alekseeva et al., 2017; Ermakov et al., 2017).\n\nAn analysis of the previous studies showed that knowledge of the role of genes in providing various mechanisms of perception is insufficient for creating a holistic picture. Certain aspects of this issue were investigated in a study conducted by (Nakagami et al., 2013) in marmosets. This results suggest that the patterns of gene expression that are potentially associated with various stages of visual cognition are different in the neurons of each layer of the visual cortex. The regulation of each neuron of primary visual cortex in marmosets is subjected to various regulating impacts when the activity of the retina changes. It should be related to a highly differentiated laminar structure of marmoset visual systems, reflecting the functions of the activity-dependent gene expression. Consequently, HTR2A gene expression depends on the level of activation of the various retinal layers.\n\nBased upon the foregoing, this study aims to examine the associations between BDNF, COMT, and HTR2A genotypes and individual characteristics of the perception of emotional visual stimuli (scenes) with distinct second-order features.\n\n\nThis study proposes the following research hypotheses\n\n1) Amplitude, spatial, and temporal characteristics of the components of visual event-related potentials in response to emotional visual stimuli (scenes) with distinct second-order features are significantly different in carriers of different BDNF, COMT, and HTR2A genotypes.\n\n2) Characteristics of attribution of emotional valences to visual stimuli (scenes) with distinct second-order features have statistically reliable difference in carriers of different BDNF, COMT, and HTR2A genotypes.\n\n\nMethods\n\nThe use of experimental subjects is in accordance with ethical guidelines as outlined in the Declaration of Helsinki. In addition, the design of the experiment, the methodological approach, the conditions of confidentiality and use of oral consent for the subjects was performed according to the Code of Ethics of Southern Federal University (SFU, Rostov-on-Don, Russia) and approved ethically by the Academic Council of the Academy of Psychology and Pedagogy of SFU, on 23 March, 2018. Oral consent for participating in research was approved since the study requires only a small amount information about the participant’s genotype, gender and age and no other additional information. It should be noted that all data were encrypted and stored in the laboratory of the Department of psychophysiology and clinical psychology and only research personal had access to it. Before the start, each participant was informed about the goal of the research, procedure, experimental conditions, and safety of their personal data.\n\nThis study involved 34 participants of both genders from 18 to 28 years (27 female), students of Southern Federal University. The study was conducted from April 27, 2018 to July 10, 2018 at the Laboratory of Psychophysiology and Experimental Psychology, Department of Psychophysiology and Clinical Psychology, Academy of Psychology and Education Sciences, Southern Federal University (Rostov-on-Don, Russia). The participants were recruited in person. Students received bonus points in their academic disciplines for their involvement in the study. All participants reported normal or corrected-to-normal vision and gave oral, informed consent before beginning experimentation. For genotype analysis buccal epithelial cells were collected from participants. Genotyping was carried out by the «Biologicheskie resheniya i tehnologii» (Moscow, Russia), where DNA was extracted from clinical material using the Proba-NK reagent kit («ООО DNK-tehnologii», Russia). Thermocycler CFX96 «Touch» (Bio-Rad, USA) was used for real-time polymerase chain reaction (PCR).\n\nFor genetic analysis were used genomic DNA preparations obtained from biological samples using DNA extraction kits with the removal of PCR inhibitors and the presence of at least 100 genomic copies in 1 μl. During the genetic examination, the following DNA sections were analyzed:\n\n– COMT catechol-O-methyltransferase gene (Genebank sequence AY341246, mutation 23753G> A, Val158Met, rs4680 code). Possible genotypes: Val/Val, Val/Met, Met/Met;\n\n– BDNF brain neurotrophic factor gene (GenBank sequence NG_011794, mutation 68690G> A Val66Met, rs6265 code). Possible genotypes: Val/Val, Val/Met, Met/Met;\n\n– HTR2A serotonin receptor gene (GenBank sequence, NG_013011, mutation 4692G> A, rs6311 (Tr2)). Possible genotypes: G/G, G/A, A/A.\n\nThe genotyping resulted in the following sample composition:\n\nCOMT gene: 9 participants (26.47 %) were carriers of Val/Val genotype, 20 (58.82%) – Val/Met and 5 (14.7 %) – Met/Met;\n\nHTR2A gene: 11 participants (32.35 %) were carriers of A/A genotype, 11 (32.35 %) – A/G and 12 (35.29 %) – G/G;\n\nBDNF gene: 23 participants (67.64 %) were carriers of Val/Val genotype and 11 (32.35%) Val/Met.\n\nFor this research, 223 images were selected from a database used in previous experiments: 85 of them are conditionally negative, 75 are neutral and 63 are positive (Ermakov et al., 2016; Ermakov et al., 2017; Stoletniy et al., 2020b; Vorobyeva et al., 2016); stimuli are available as Extended data (Kovsh & Yavna, 2020). To obtain stimuli with corresponding characteristics, the computer model of the second-order visual mechanisms developed by (Babenko & Yavna, 2018) was applied. Figure 1 below demonstrates examples of original images and the results of their processing by the model. Stimuli images were generated from contrast modulations in the carrier frequency range from 4 to 32 cycle per image. Resolution of pictures is 1024х768 (slightly more 16°х12° of visual angle on the 100 cm distance). The average luminance of the generated images was equal to 110 of 256 greyscale (30 cd/m2). The background brightness was adjusted to the same level. All 223 selected images were processed and then organized in a stimulus sequence.\n\nParticipants were seated in good lit room, facing a computer screen, at viewing distance of 100 cm. A grey background (30 cd/m2) was on the monitor all the time of trial, with and without stimuli. Stimulus image was presented in center of screen and its duration was 500 ms. The interstimuli interval was 500–1500 ms (Figure 2). Stimulus pictures were presented in random order regardless of their emotional valence. Participants were asked to examine the stimuli in terms of what emotions this images evokes in them. When evaluating the stimulus as negative, it was necessary to press the key 1 on the keyboard for negative assessment, 2 for neutral, 3 for positive. The next image appeared on the screen only after the subject evaluated the previous one and the interval between stimuli was completed. Furthermore, participants were instructed to blink only when stimulus ended.\n\nThe frequency of responses was analyzed using the REdaS Package for R, version 0.9.3., in order to calculate confidence intervals for average response rate with α = 0.05 (Maier, 2015).\n\nA Neurovisor-136 multi-channel EEG system («Medical Computer Systems», Moscow, Russia) was used to record the EEG from 128 electrodes mounted on elastic cap and referenced on the left and right ears. Electrodes were placed according to 10–5 system (Oostenveld & Praamstra, 2001). EEG signal was digitalized at 1000 Hz, and a bandpass filter of 0.5–50 Hz was applied.\n\nThe ERPs were processed using the EEGLAB version 4.11 – an interactive Matlab/Octave toolbox for processing electrophysiological data (Delorme & Makeig, 2004). Before averaging the ERPs, the recordings were filtered and the epochs with eye blinks and other artifact of non-brain origin were rejected. The ERPs were averaged 100 ms before the stimulus onset and 500 ms after it. Baseline was corrected based on 100 ms pre-stimulus period. ERPs were computed and grouped according to type of response that participants gave – negative, neutral and positive and theirs genotype. The ERPs grouped by type of emotional assessment were compared separately for COMT, BDNF, HTR2A samples (For example: COMT x Negative evaluation x Val/Val vs Val/ Met; Val/Val vs Met/Met; Val/Met vs Met\\Met etc.). Comparisons were carried out using an unpaired Student’s T-test with p < 0.05. ERPs and topography figures were conducted in EEGLAB. The results from all ERP stimuli are available as Underlying data (Stoletniy et al., 2020a).\n\n\nResults\n\nThis analysis showed that there was no statistically reliable difference in the average response rate in participants with different BDNF gene genotypes regardless type of assessment of target images (Figure 3, upper line). Nevertheless, it should be noted that the ratio of the average number of choices of neutral responses is the reverse of that when choosing negative and positive judgments; the individual participants with the Val/Val genotype chose the neutral response slightly more often than the participants with the Val/Met genotype.\n\nMM- Met/Met genotype, VM – Val/Met, VV- Val/Val for COMT and BDNF genes; AA – A/A, AG – A/G, GG – G/G for HTR2A gene.\n\nParticipants with different COMT genotypes chose negative assessments with an average frequency (about 32–33%). There were no significant differences in the frequency of negative responses in the groups. Nevertheless, the distribution of neutral and positive responses is of more interest (Figure 3, middle line). On the one hand, participants with the Val/Val and Val/Met genotype chose the positive assessment of stimuli significantly more often (19% and 13%, respectively) than carriers of Met/Met. On the other hand, the carriers of the Met/Met genotype were significantly often (18% and 13% compared to Val/Val and Val/Met groups) chose to evaluate the images as neutral. There were no significant differences between the average frequency of choosing the neutral and positive responses between the Val/Met and Val/Val groups.\n\nCarriers of different HTR2A genotypes chose negative responses with different average frequency but no statistically reliable difference, because the confidence intervals do not intersect. Although the histogram has a stepwise character from a higher average frequency for carriers of the A/A genotype (39%) to lower frequency for carriers of the A/G (33%) and G/G (28%) genotype (Figure 3, bottom line). A more interesting situation has developed for the neutral and positive types of responses. Participants with the G/G genotype were significantly more likely (46%) to choose the neutral response when evaluating experimental stimuli than those with the A/A (25%) and A/G (29%) genotypes. On the contrary, the G/G group had chosen positive assessment less often (26%), than A/A (37%) and A/G (37%) groups. There was no statistically reliable difference between the average frequency of choosing the neutral and positive responses between the A/A and A/G groups.\n\nBDNF. The comparison of event-related potentials when choosing the negative assessment in the groups of carriers of Val/Met and Val/Val genotypes showed significant differences in the amplitude of the event-related response (p <0.003, t = -3.5) in the time range of 150–230 ms (Figure 4a). As can be clearly seen in the figure, the amplitude of the P2 component of the ERP recorded for negative response in participants with the Val/Val genotype is significantly higher than that of the Val/Met group. When choosing the neutral assessment statistically significant differences was found in latency of 172–227 ms (Figure 4b). The ERP amplitude in the group of carriers of the Val/Val genotype is significantly higher (p < 0.02, t = -1.22) than that of the Val/Met group. Choose of positive response to the stimuli elicited an increase in amplitude of ERP peak in the latency of 133–230 ms. ERP peak for the Val/Val group was significantly higher (p < 0.029, t = -2.36) than that of the Val/Met group (Figure 4c). Table 1 presents the values of the ERPs amplitudes in Oz electrode at a latency of 200 ms for the BDNF group.\n\nERP for the groups with Val/Met and Val/Val BDNF genotypes in Oz electrode when choosing the negative (a), neutral (b) or positive (c) assessment of stimuli. The gray bar indicates the latency with statistically significant differences (p < 0.05).\n\nThe topography of the potentials distribution shows that the choice of the negative response caused activation in the occipital regions of the cortex. Moreover, it was higher in the group of carriers of the Val/Val genotype of the BDNF gene (Figure 5a). The choice of the neutral assessment caused activation in the occipital regions of the cortex to a greater extent in the group of carriers of the Val/Val genotype (Figure 5b). Nevertheless, activity in the anterior and central areas of the cerebral cortex is somewhat greater in the group of participants with the Val/Met genotype of the BDNF gene. Selection of the positive assessment caused an increase in the amplitude of positive peaks in the region of 140–230 ms in the occipital regions of the cortex, to a greater extent in carriers of the Val/Val genotype of the BDNF gene, as in both previous cases (Figure 5c).\n\nTopography of the distribution of potentials at a latency of 200 ms (where statistically significant differences were found) from onset of stimuli when choosing negative (a), neutral (b) or positive (c) assessment for the Val/Met and Val/Val BDNF genotypes.\n\nCOMT. The comparison of ERPs in the groups of participants with the Val/Met and Val/Val genotypes did not show significant differences regardless of assessment type: for negative, p < 0.94, t = -0.06; for neutral, p < 0.59, t = -1.7; for positive, p <0.95, t = -0.10 (Figure 6a–c, solid and intermittent waves on the figures). On the contrary, amplitude of the event-related potentials in participants with the Met/Met genotype is significantly lower compared to Val/Val and Val/Met groups during every type of assessment (Figure 6, a, b, c, dotted wave). For negative evaluation this differences were observed in latency to the 110 to 175 ms (Val/Met - p < 0.03, t = -2.33; Val/Val - p < 0.03, t = 0.6), for neutral in 120 to 170 ms (Val/Met - p < 0.006, t = -3.10, Val/Val - p < 0.03, t = 2.19), for positive - 135 to 155 ms (Val/Met - p <0.02, t = -1.39, Val/Val - p <0.03, t = -2.39). It should be noted that the amplitude of the event-related potential for Met/Met is higher when participants assessed stimuli as the neutral and positive, then negative ones. Table 1 presents the values of the ERPs amplitudes in Oz electrode at a latency of 150 ms for the COMT group.\n\nERP for the groups with Val/Met, Val/Val and Met/Met COMT genotypes in Oz electrode when choosing the negative (a), neutral (b) or positive (c) assessment of stimuli. The gray bar indicates the latency with statistically significant differences (p < 0.05). Significant differences between ERPs in Val/Val and Val/Met groups were not found.\n\nTopography of potentials clearly shows a decrease in activity of occipital regions in the Met/Met group in latency of 150 ms comparing to Val/Met and Val/Val groups. On the contrary Val/Met and Val/Val group showed increase in activity also in the occipital regions of the brain (Figure 7a–c).\n\nTopography of the distribution of potentials at a latency of 150 ms (where statistically significant differences were found) from onset of stimuli when choosing negative (a), neutral (b) or positive (c) assessment for the Met/Met, Val/Met and Val/Val COMT genotypes.\n\nHTR2A. Comparing the ERPs of A/G and G/G genotype groups showed no statistical difference between them, regardless of assessment type – negative (p < 0.17, t = 1.41), neutral (p < 0.56, t = -0.59) or positive (p < 0.84, t = -0.19). At the same time, chose a negative response type demonstrated that the amplitude of ERP in carriers of the A/A genotype was significantly different from such of the A/G genotype (p < 0.03, t = 2.29) and G / G (p < 0.04, t = 3.12) in the range of 117–133 ms from the onset of stimulus (Figure 8a). Choosing neutral response by carriers of A/A genotype reflected in significant decrease of ERP component’s amplitude in 350–370 ms latency compared to A/G (p < 0.02, t = -2.47) and G/G (p < 0.03, t = -2.25) groups (Figure 8b). Similar to ERP comparison for negative assessment, evaluating stimuli as positive by carriers A/A genotype showed significant increase of ERP amplitude in latency 105 to 124 ms compared to such in A/G (p < 0.01, t = 2.72) and G/G (p <0.03, t = 2.32) genotype carriers (Figure 8c). Table 1 presents the values of the ERPs amplitudes in Oz, Po7 and Poz electrodes at a latency of 120 and 360 ms for the HTR2A group.\n\nERP for groups with different HTR2A genotypes in Oz, Po7 and Poz electrodes when choosing negative (a), neutral (b) or positive (c) assessment respectively. The gray bar indicates the latency with statistically significant differences (p < 0.05). Significant differences between ERPs in A/G and G/G groups were not found.\n\nThe topography of the distribution of potentials shows a high positivity when choosing negative assessment mainly in the occipital regions of the A/A group and a tendency to decrease of it in same regions in the group of carriers of the A/G genotype, and to a greater extent – in carriers of the G/G genotype (Figure 9a). In the neutral response situation ERP amplitude distribution in carriers of the A/A genotype is somewhat homogeneous over the entire surface of the scalp. However, in carriers of the A/G and G/G genotypes it is much greater in the occipital and parietal regions (Figure 9b). When choosing the positive response, high-amplitude ERPs are localized in the occipital regions. Moreover, the activity in the A/A group is most higher, to a lesser extent it is expressed in the A/G group, and even lower in G/G groups (Figure 9c).\n\nTopography of the distribution of potentials at a latency of 120 ms from onset of stimuli (where statistically significant differences were found) when choosing negative (a) or positive (c) and 360 ms when choosing neutral (b) assessment for the A/A, A/G and G/G HTR2A genotypes.\n\n\nDiscussion\n\nAs shown in the Results section, the BDNF Val/Val genotype is associated with a predominance of neutral assessment of visual stimuli processed by second-order filters. Neutral, positive, and negative judgments of visual stimuli are associated with a significantly higher positivity of the P2 component in the occipital electrodes, which may be explained by the fact that compared to the carriers of BDNF Val/Met genotype the carriers of the BDNF Val/Val genotype consumed large brain resources for recognition and differentiation of stimuli, experiencing difficulties in the implementation of these processes. Moreover, brain activity in the anterior and central areas is more pronounced in carriers of the Val/Met genotype. We obtained similar results in previous series of the experiment (during recognition of unprocessed visual stimuli), which may indicate that the Met allele of the BDNF gene is associated with more efficient processes of recognition of emotional scenes (Ermakov et al., 2017). This feature can be explained by a more intense activation of the structures of the limbic system (Vorobyeva et al., 2019) and stronger functional cortico-limbic bonds (Loewenstern et al., 2019), activated during recognition of facial expression in carriers of the Met allele of the BDNF gene.\n\nAccording to our results, when judging the stimulus as negative, neutral, or positive negativity was reliably expressed in 110–175 ms (i.e., there is a higher, more negative amplitude of the N170 component) in the occipital electrodes in carriers of the COMT Met/Met genotype.\n\nThis aspect may be related to the fact that, when evaluating scenes with distinct second-order features, the carriers of the COMT Met/Met genotype primarily pay attention to the facial expression of individuals depicted in the stimulus pictures and assess the emotional valence of the stimulus based on these data (Eimer & Holmes, 2007). However, this process consumes more brain resources to recognize faces emotional expression.\n\nAccording to a number of studies, carriers of the COMT Met/Met genotype have higher indices of emotional intelligence (Kosonogov et al., 2019), more successfully differentiate and express emotions (Gohier et al., 2014a). Carriers of the COMT Met/Met genotype more decently respond to positive stimuli. The Val/Val genotype is associated with less intense feelings in negative experience, i.e. their level of life satisfaction is less dependent on external events (Kovalenko & Pavlenko, 2009; Shield et al., 2004)\n\nThe HTR2A G/G genotype is associated with a significantly more frequent evaluation of stimuli with distinct second-order features as neutral ones, as well as with a significantly rarer assessed of stimuli as positive ones. The A/A genotype is associated with a significantly higher amplitude of the P1 peak, recorded mainly in the occipital regions of the brain when evaluating visual stimuli processed by second-order filters as positive or negative ones; with a significantly lower wave amplitude in the range of 350–370 ms when assessed as emotionally neutral. These results may indicate that carriers of A/A genotype consume more brain resources at the stage of distinguishing of a significant stimulus among insignificant ones, experiencing difficulties in the implementation of this process (difficulties in pre-attentive processing; component P1 is associated with selectivity of attention), and less resources are consumed duding categorization and conscious evaluation of the emotional content of stimuli (Kovalenko & Pavlenko, 2009; Polich, 2012).\n\n\nConclusions\n\nThe analysis of the electrical activity of the brain in carriers of various genotypes of the BDNF, COMT, and HTR2A genes when choosing different types of responses in attributing emotional valences to visual scenes processed by the filters with the distinct second-order features enabled us to draw the following conclusions.\n\n1) In carriers of the Val/Val genotype the amplitude of the P2 component of visual event-related potentials in the occipital regions of the brain is higher than in carriers of the BDNF Val/Met genotype, regardless of the type of response chosen when evaluating the presented images.\n\n2) The highest ERP amplitude of the three possible assessments (positive, negative, neutral) was found in the response recorded in the neutral evaluation of the presented stimuli; this fact is valid for carriers of the BDNF Val/Val and Val/Met genotypes.\n\n3) Regardless of the type of stimuli, the participants chose responses of different modalities with approximately the same frequency.\n\n1) In the group of participants with the COMT Met/Met genotype for all the three types of responses (regardless the stimuli images) the induced brain activity is characterized by a significant increase in negativity in peak amplitude latency from 120 to 170 ms and occurs mainly in the occipital cortex brain.\n\n2) When carriers of Met/Met assessed the stimuli as negative the amplitude of this early ERP component is showed increasing negativity, compared with that when choosing neutral or positive responses.\n\n3) Met/Met genotype carriers statistically reliably chose neutral assessment more often and positive less often than carriers of other two genotypes.\n\n1) There were no statistically significant differences between the ERP waves in carriers of the HTR2A A/G and G/G genotypes, regardless of the choice of the emotional valences of responses. On the contrary, A/A group had statistically reliable differences in ERPs peaks compared to A/G and G/G genotypes carriers.\n\n2) When choosing negative and positive assessment, carriers of A/A genotype had statistically reliable differences in early latency peak that had large amplitude, compared to A/G and G/G genotypes carriers. Contrariwise, in neutral assessment situation, a statistically reliable differences in ERPs shifted to later positive peak (around 360 ms) that had an increasing negativity in participants with A/A genotype.\n\n3) The localization of activity also has differences in carriers of different HTR2A genotypes. When choosing negative and positive responses, the topography of significant high-amplitude components of the ERP shows their localization in the occipital regions; with a late latent significant ERP when choosing a neutral type of responses, increased activity also affects the parietal areas (which is more reliable to carriers of the A/G and G/G genotypes compared to carriers of the A/A genotype).\n\n4) G/G genotype carriers were significantly more often chose neutral response and less often positive response for stimuli than carriers of other two genotypes.\n\nIn this study, associations between the BDNF, COMT, and HTR2A genotypes and individual characteristics of the perception of emotional visual stimuli with distinct second-order features were investigated. The findings of the study confirmed both hypotheses of the research. Furthermore, the results lead to conclusion that the images preserving second-order features, where all the homogeneous areas are removed, contain all the necessary information for their successful recognition. This may be explained by the work of autonomous mechanisms operating at a pre-attentive stage of visual processing (Babenko & Yavna, 2018). At the same time, various genotypes are associated with varying degrees of success of the pre-attentive and conscious stages of processing emotional visual stimuli (scenes), which confirms the hypothesis that there are genetic factors that individualize the mechanisms of visual perception.\n\n\nData availability\n\nOpen Science Framework: EEGLAB datasets for study of event-related potentials during categorization of emotionally-charged and neutral images with distinct second-order features in carriers of polymorphisms of the COMT, HTR2A, BDNF genes. https://doi.org/10.17605/OSF.IO/PDHGZ (Stoletniy et al., 2020a).\n\nThis project contains the follow underlying data:\n\n1_Negative estimation (ERP datasets recorded when participant chose negative assessment of stimuli).\n\n2_Neutral estimation (ERP datasets recorded when participant chose neutral assessment of stimuli).\n\n3_Positive estimation (ERP datasets recorded when participant chose positive assessment of stimuli).\n\nData Characteristics.xlsx (detailed description of datasets files).\n\nData Description.docx (detailed description of experiment condition and datasets).\n\nOpen Science Framework: Stimuli database for studying emotional assessment of emotionally-charged images with distinct second-order features. https://doi.org/10.17605/OSF.IO/WY2VF (Kovsh & Yavna, 2020).\n\nThis project contains all visual stimuli provided for this study, alongside a data description list of the image features.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAftanas LI, Varlamov AA, Pavlov SV, et al.: Affective picture processing: Event-related synchronization within individually defined human theta band is modulated by valence dimension. 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PubMed Abstract \| Publisher Full Text\n\nStoletniy A, Kovsh E, Vorobyeva E: EEGLAB datasets for study of event-related potentials during categorization of emotionally-charged and neutral images with distinct second-order features in carriers of polymorphisms of the COMT, HTR2A, BDNF genes. 2020a. http://www.doi.org/10.17605/OSF.IO/PDHGZ\n\nStoletniy A, Kovsh E, Yavna D: Stimul database for studying emotional assessment of emotionally-charged images. 2020b. http://www.doi.org/10.17605/OSF.IO/ZYG54\n\nTamm G, Kreegipuu K, Harro J: Perception of emotion in facial stimuli: The interaction of ADRA2A and COMT genotypes, and sex. Prog Neuropsychopharmacol Biol Psychiatry. 2016; 64: 87–95. PubMed Abstract \| Publisher Full Text\n\nVorobyeva E, Hakunova F, Skirtach I, et al.: A review of current research on genetic factors associated with the functioning of the perceptual and emotional systems of the brain. SHS Web of Conferences. 2019; 70: 09009. Publisher Full Text\n\nVorobyeva E, Kovsh E, Yavna D: Visual evoked potentials elicited by culturally-specific images in women with different levels of hostility. Int J Psychophysiol. 2016; 108: 69. Publisher Full Text\n\nWeiss EM, Freudenthaler HH, Fink A, et al.: Differential Influence of 5-HTTLPR - Polymorphism and COMT Val158Met—Polymorphism on Emotion Perception and Regulation in Healthy Women. J Int Neuropsychol Soc. 2014; 20(5): 516–524. PubMed Abstract \| Publisher Full Text\n\nWilson HR, Gelb DJ: Modified line-element theory for spatial-frequency and width discrimination. J Opt Soc Am A. 1984; 1(1): 124–131. PubMed Abstract \| Publisher Full Text\n\nYavna D, Babenko V: Psychophysical and psychophysiological evidence for specificity of second-order visual mechanisms [Psihofizicheskie i psihofiziologicheskie dokazatel’stva specifichnosti zritel’nyh mehanizmov vtorogo porjadka]. Proceedings of the 16th International Conference on Neurocybernetics. 2012; 412–415.\n\nYavna D, Babenko V, Soloviev A: Cortical localization of second-order visual mechanisms. 41(Perception. Supplement. 35th European Conference on Visual Perception (2-6 September, Alghero, Italy). Abstracts.): 2012; 249." }	[ { "id": "72768", "date": "16 Dec 2020", "name": "Elena Samoylenko", "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\nWhy were COMT, BDNF and HTR2A genes selected for the study, although the article deals with topic of visual perception?\n\nWhy was the analysis approach with intragroup comparison for one gene chosen, and not comparison for combinations of all genes genotypes?\n\nEEG and ERP were recorded in 128 scalp electrodes. You did not indicate if you applied correction for multiple comparisons.\n\nSample of participants is sufficient for ERP study, but small enough for genetic research. Please add “Limitation” section in the article and describe confines of research due to relatively small sample size.\n\nPlease unify the designation of the ERP components - either P1 or P100 type of 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?\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": "84878", "date": "21 May 2021", "name": "Talis Bachmann", "expertise": [ "Reviewer Expertise Experimental psychology", "psychophysiology." ], "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 language of this paper is extremely poor both in terms of grammar and idiomatic aspects. This makes it very difficult to smoothly read the text and get precisely to what the authors want to say. In many places sentences do not make sense or are impossible to understand unambiguously: e.g., p 4 left column, middle (\"Carriers of the COMT Met allele...\"), p 4 right column, middle (\"An analysis of the previous...\"), p 6, left column, middle (\"The ERPs were averaged 100 ms before...\"), etc.\n\nIn many places there are no references to the sources of statements about factual knowledge from earlier research (e.g., p 3 bottom left, 3rd para from bottom right).\n\nThe buildup from Introduction to hypotheses is poor and unfocused. Why study specifically these genes in light of the set tasks and hypotheses? Theoretical introduction is eclectic and does not clearly lead to specific hypotheses. It is never mentioned that the study is essentially exploratory.\n\nThe description of the method is cryptic in several places and terms used are too often vague, unspecified and different from what is currently used in the worldwide publications on the topic. Page 5, middle: \"... stimuli with corresponding characteristics...\" -- what does this precisely mean? Few lines further down: \"Resolution of pictures is 1024x768 (slightly more 16degx12deg of visual angle...\" -- display resolution is lumped with visual angle of the screen. Further down, \"The next image appeared on the screen only after the subject evaluated the previous one and the interval between stimuli was completed\" contradicts the earlier description of the trial timing. 'Second order mechanisms' are used instead of, e.g., spatially filtered original images. Page 6, Results: first sentence of the Behavioral results -- 'average response rate' is not sufficiently clear what is meant by this. The design is difficult to understand because on the one hand there are pictures with different emotional connotations, on the other hand there are different response options. How these are interrelated is difficult to comprehend.\n\nSome aspects of the method are questionable. For example, the frequency of presentation of stimuli with different affective categories is unequal, possibly leading to artefacts. Value and arousal dimensions of pictures are not controlled and counterbalanced or cross-varied. The time intervals between successive stimulation presentations may be too short. (For instance, readiness potentials, CNVs and evoked potentials may be mutually cancelled or noised. There is no clear P300 visible, which is suspect for data analysis.) The conclusion \"Regardless of the type of stimuli...\" (p 14, 3)) hints at the possibility that there is something wrong in the design.\n\nConclusions in the Discussion and at the end can not be derived from the carried-out study as if they are firmly supported.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/9-1010
https://f1000research.com/articles/9-287/v1	24 Apr 20	{ "type": "Research Article", "title": "Anti-retroviral therapy after “Treat All” in Harare, Zimbabwe: What are the changes in uptake, time to initiation and retention?", "authors": [ "Takura Matare", "Hemant Deepak Shewade", "Ronald T. Ncube", "Kudzai Masunda", "Innocent Mukeredzi", "Kudakwashe C. Takarinda", "Janet Dzangare", "Gloria Gonese", "Bekezela B. Khabo", "Regis C. Choto", "Tsitsi Apollo", "Ronald T. Ncube", "Kudzai Masunda", "Innocent Mukeredzi", "Kudakwashe C. Takarinda", "Janet Dzangare", "Gloria Gonese", "Bekezela B. Khabo", "Regis C. Choto", "Tsitsi Apollo" ], "abstract": "Background: In Zimbabwe, Harare was the first province to implement “Treat All” for people living with human immunodeficiency virus (PLHIV). Since its roll out in July 2016, no study has been conducted to assess the changes in key programme indicators. We compared antiretroviral therapy (ART) uptake, time to ART initiation from diagnosis, and retention before and during “Treat All”. Methods: We conducted an ecological study to assess ART uptake among all PLHIV newly diagnosed before and during “Treat All”. We conducted a cohort study to assess time to ART initiation and retention in care among all PLHIV newly initiated on ART from all electronic patient management system-supported sites (n=50) before and during “Treat All”. Results: ART uptake increased from 65% (n=4619) by the end of quarter one, 2014 to 85% (n=5152) by the end of quarter four, 2018. A cohort of 2289 PLHIV were newly initiated on ART before (April-June 2015) and 1682 during “Treat all” (April-June 2017). Their age and gender distribution was similar. The proportion of PLHIV in early stages of disease was significantly higher during “Treat all” (73.2% vs. 55.6%, p<0.001). The median time to ART initiation was significantly lower during “Treat All” (31 vs. 88 days, p<0.001). Cummulative retention at three, six and 12 months was consistently lower during “Treat all” and was significant at six months (74.9% vs.78.1% p=0.022). Conclusion: Although there were benefits of early ART initiation during “Treat All”, the programme should consider strategies to improve retention.", "keywords": [ "ART outcomes", "test and treat", "universal test and treat", "time to treatment", "HIV", "SORT IT", "Operational research" ], "content": "Introduction\n\nGlobally in 2018, there were an estimated 37.9 million people living with human immunodeficiency virus (PLHIV)1. The majority of them (54%) are in eastern and southern Africa. Since the start of the epidemic, 32 million people were estimated to have died from acquired immune deficiency syndrome related illnesses1. An estimated 23 million people are accessing antiretroviral therapy (ART) globally1.\n\nFollowing evidence from research studies on the clinical and public health benefits of immediate ART, in July 2015, the World Health Organization (WHO) released guidelines on when to start ART and pre-exposure prophylaxis for HIV2,3. These guidelines recommended ART to be offered to all PLHIV (“Treat All”), regardless of CD4 threshold and/or WHO clinical stage. In July 2018, WHO reported that 84% of low- and middle-income countries had adopted the “Treat All” policy4. Research in other countries demonstrated that after implementation of “Treat All”, people on ART had better outcomes2,5–8.\n\nZimbabwe has a generalized HIV epidemic, with an estimated 1.3 million PLHIV and an HIV prevalence of 14% among adults (15-64 years)9–11. In July 2016, Harare province was the first to start implementing “Treat All”12. Since the roll out of “Treat All”, no comparative study has been conducted to measure the changes in linkage to care and ART outcomes. We therefore assessed ART uptake, time to ART initiation from diagnosis and retention among PLHIV before and during “Treat All” in Harare.\n\n\nMethods\n\nFor ART uptake, we used an ecological design involving aggregate secondary programme data and all PLHIV newly diagnosed before (2014 to June 2016) and during “Treat All” (July 2016 to 2018) in Harare were the study population.\n\nFor time to ART initiation and retention, we used a cohort design involving patient wise secondary programme data. We included all PLHIV newly initiated on ART from 50 electronic patient management system (ePMS) sites in Harare before (April-June 2015) and during “Treat All” (April-June 2017).\n\nGeneral setting. Harare province constitutes 16.3% of the population in Zimbabwe9, with an estimated 0.2 million PLHIV, the highest among all the provinces10. It has the highest number of patients active on ART, with 77 ART sites providing HIV diagnosis and treatment. As on October 2018, the 50 ePMS sites constituted 52% of all people on ART in Harare province.\n\nART initiation before “Treat All”. Prior to “Treat All”, PLHIV were initiated on ART based on a CD4 eligibility criteria of <500 cells/mm3 (with priority given to those <300 cells/mm3 OR WHO clinical stage 3 or 4). Additionally, pregnant and/or breast feeding women, sero-discordant couples, Hepatitis B virus co-infection, people diagnosed with tuberculosis (TB) and children ≤5 years were also eligible irrespective of CD4 count.\n\nBaseline investigations at primary level of care included urine dipstick (glucose, protein), haemoglobin, CD4 count for immunologic staging, cryptococcal antigen screen for adults with CD4 count <100 cells/mm3, screen for pregnancy, syphilis if 12 years or older and hepatitis B infection13. Other investigations included full blood count, creatinine and hepatitis C serology at secondary or tertiary levels of care. PLHIV were screened for active TB and other Opportunistic Infections (OIs)13. If active TB was diagnosed, ART was initiated within 2–8 weeks of initiation of anti-TB treatment and within two weeks for advanced TB disease.\n\nPatients were followed up monthly initially and then every three months. Viral load testing targeted those suspected of HIV treatment failure14. A patient with confirmed virologic, immunologic or clinical failure were switched to second line ART13. Data were routinely recorded in the ART register, patient OI/ART care booklet. The patient level data was also routinely captured electronically in the 50 ePMS sites.\n\nChanges in ART initiation and further management during “Treat All”. All PLHIV are eligible for ART regardless of CD4 count or WHO clinical staging12. PLHIV are monitored for viral load at six months after starting ART, 12 months then annually thereafter if stable13.\n\nFor ART uptake, we extracted quarterly aggregate number of new HIV diagnoses and new ART initiations from the District Health Information System 2 (DHIS 2). For time to ART initiation and retention, the source of patient level data was the ePMS and included OI/ART number; ART site; date of HIV diagnosis; date of ART initiation; date of birth; baseline characteristics and outcome at three, six and 12 months - alive and on treatment, death, loss to follow up, stopped ART, transferred out; and date of ART outcome. Operational definition of outcomes has been depicted in Table 1.\n\nHIV: human immunodeficiency virus; PLHIV: people living with HIV; ART: antiretroviral therapy; ePMS: electronic patient management system.\n\nFor ART uptake, we calculated quarterly proportions of newly diagnosed PLHIV initiated on ART (Microsoft Excel 2010, Microsoft, Redmond, WA, USA) and presented them using bar diagram and trend lines.\n\nWe summarized time to ART initiation after diagnosis using median (interquartile range - IQR) and compared it before and during “Treat All” using Mann Whitney U test. We also used frequency and proportions to summarize time to ART (same day, 2–7 days, 8–14, 15–30 days, 31–90 days, 91–180 days, >180 days] and compared it before and during “Treat All” using z test for proportions. Baseline characteristics and cumulative retention in care were also compared using z test for proportions (STATA - version 12.1, copyright 1985–2011 Stata Corp LP USA).\n\nWe obtained ethics approval from the Union Ethics Advisory Group (EAG), Paris, France (EAG number: 47/19, 29 April 2019) and the Medical Research Council of Zimbabwe (MRCZ), Harare (MRCZ/E/245, 19 August 2019). As this study involved review of existing programme records, we sought and obtained waiver of written informed consent from the ethics committees. Secondary data were extracted after obtaining administrative approval from the concerned authorities.\n\n\nResults\n\nA total of 84,776 people were diagnosed with HIV before (2014 to June 2016) and 82,672 during “Treat All” (July 2016 to 2018). ART uptake increased from 65% (n=4619) by the end of quarter one, 2014 to 85% (n=5152) by the end of quarter four, 2018. Starting quarter three, 2016 (implementation of “Treat All”), there was an increasing trend in the new ART initiations, as well as the ART uptake up to quarter two, 2017. Between quarter three, 2017 and quarter four, 2018, there was an increasing trend in ART uptake, which peaked to 85% (n=5152) (Figure 1). However, during this period, the absolute number of HIV diagnoses as well as the absolute new ART initiations reduced.\n\nPLHIV: People Living with Human Immunodeficiency Virus; ART: Antiretroviral therapy\n\n^aggregate numbers for each quarter were extracted to calculate ART uptake, source of data is district health information system (DHIS-2)\n\n” Treat All” means all individuals with confirmed HIV diagnosis are eligible for ART irrespective of WHO clinical stage or CD4 count.\n\n#During 2014, the CD4 count eligibility criteria was raised from <350 to <500 cells/mm33. As more people in pre-ART care were eligible for ART, there was an increase in new ART initiations which resulted in corresponding increase in ART uptake. Similarly, there was an increase in new ART initiations which resulted in corresponding increase in ART uptake after “Treat All”.\n\nA total of 2289 PLHIV were newly initiated on ART before (April-June 2015) and 1682 were newly initiated during “Treat all” (April-June 2017). The median age (in years) for the two groups was similar, 31 (IQR: 27, 41). Gender distribution before and during “Treat all” was also similar (women: 58.6% vs. 58.3% respectively). The proportion of people in early stages of disease (WHO clinical stage I or II) was significantly higher during “Treat all” (73.2% vs. 55.6%, p<0.001).\n\nWe were not able to calculate the time interval for 41% of people because of missing dates. Of those with dates available, the median time to ART initiation from diagnosis was significantly lower during “Treat All” when compared to before “Treat all” (31 vs. 88 days, p<0.001). ART initiation on the same day (19.3% vs. 4.9%, p=<0.001) and within 14 days (25.5% vs 12.5%, p<0.001) was significantly higher during “Treat All” (Table 2).\n\nPLHIV: people living with human immunodeficiency virus; ART: antiretroviral therapy; ePMS: electronic patient management system\n\n”Treat All” means all individuals with confirmed HIV diagnosis are eligible for ART irrespective of WHO clinical stage or CD4 count.\n\n^As on October 2018, the 50 ePMS sites constituted 52% of all people on ART in Harare province.\n\nRetention at three, six and 12 months was consistently lower during “Treat All” compared to before “Treat All”. It was significantly lower at six months (74.9% vs.78.1% p=0.022) (Table 3).\n\nPLHIV: people living with human immunodeficiency virus; ART: antiretroviral therapy; ePMS: electronic patient management system; LFTU: loss to follow up\n\n*” Treat All” means all individuals with confirmed HIV diagnosis are eligible for ART irrespective of WHO clinical stage or CD4 count.\n\n^As on October 2018, the 50 ePMS sites constituted 52% of all people on ART in Harare province.\n\n#z test of proportions (proportion retained in care)\n\n\nDiscussion\n\nThis is the first study in Zimbabwe that attempts to document the changes in ART initiation and retention before and during “Treat All”. There were three programme relevant findings.\n\nFirst, ART uptake improved immediately after the implementation of “Treat All” (starting July 201612). When compared to quarter one, 2014 (65%), ART uptake improved in quarter four, 2018 (85%). However, there was a drop in the number of PLHIV diagnosed and number of new ART initiations from quarter three, 2017 to quarter four, 2018. The improvement in uptake during this period was not due to increase in annual new ART initiations. It was because of faster falling trends of HIV diagnoses relative to the falling trends of new ART initiations. Hence, the programme should explore the reasons for reduction in new ART initiations and take necessary action to ensure that it moves closer to the second ‘90’ of UNAIDS 90-90-90 targets by 202015.\n\nSecond, time to ART initiation significantly reduced during “Treat All” when compared to before “Treat All” (31 vs. 88 days). Though the median time reduced, the proportion initiated on ART on the day of diagnosis was less compared to 65% in mission hospitals of Zimbabwe in 201716. Therefore there is further opportunity to reduce time to linkage to ART. Faster linkage is known to be associated with better retention17.\n\nThird, retention did not improve despite more PLHIV being clinically asymptomatic at ART initiation during “Treat All” when compared to before “Treat All”. This was contrary to findings in Malawi, where retention at 12 months during “Treat All” (83%) was higher than before “Treat All” (76%)8. In Kenya and Uganda (2017), retention at one year was 89%17. In mission hospitals of Zimbabwe (2017), retention at three months was as high as 90%16. Good health (at diagnosis) has been reported to act both as a barrier as well as a facilitator to ART initiation18,19, therefore, we speculate that good health at ART initiation may also act both as a barrier as well as a facilitator to ART retention. Another potential reason for the observed lower retention could be deficiencies in coverage of quality adherence counselling with likely increased work load during the “Treat All” era.\n\nThe programme may consider combination intervention strategy to improve linkages to care and retention. This strategy includes i) point-of-care CD4 testing at the time of diagnosis, ii) accelerated ART initiation, and iii) short message service (SMS) health messages and appointment reminder20.\n\nThe strength of this study were the large sample size, which included all sites in Harare reporting through DHIS 2 and ePMS. There were however some limitations. First, inherent to observational studies is the possibility of documentation errors that could not be validated. Second, some baseline characteristics namely; body mass index, CD4 count, anaemia and Hepatitis B and C co-infections were incomplete in at least 80% of the records and were excluded from analysis.\n\nIn conclusion, as expected, “Treat All” increased ART uptake and reduced time to ART initiation. Retention in care did not improve as a result of “Treat all”. This is a clarion call for the programme to focus interventions on efficient linkage to ART and retention in care in order to reap the benefits of “Treat All”.\n\n\nData availability\n\nFigshare: Matare T et study 2020 ART uptake, initiation delay and retention data, https://doi.org/10.6084/m9.figshare.c.494439921.\n\nThis project contains the following underlying data:\n\n- ART initiation delay and retention data: Patient wise ART initiation delay and retention data of people initiated on ART before (Q2-2015) and during (Q2-2017) \"Treat All\" from the 50 ePMS sites in Harare, Zimbabwe.\n\n- ART uptake data: Aggregate quarterwise ART uptake data from 2014 to 2018 in Harare.\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\nUNAIDS: Global HIV statistics-Fact Sheet. Geneva, Switzerland. 2019. Reference Source\n\nEholié SP, Badje A, Kouame GM, et al.: Antiretroviral treatment regardless of CD4 count: the universal answer to a contextual question. AIDS Res Ther. 2016; 13: 27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization (WHO): Guidelines Guideline on When To Start Antiretroviral Therapy and on Pre-Exposure Prophylaxis for HIV. Harare, Zimbabwe. 2015; 78. Reference Source\n\nWorld Health Organization (WHO): Who HIV Policy Adoption and Implementation Status in Countries. Geneva, Switzerland. 2018; 2019. Reference Source\n\nKimmel AD, Bono RS, Keiser O, et al.: Mathematical modelling to inform “treat all” implementation in sub-Saharan Africa: a scoping review. J Virus Erad. 2018; 4(Suppl 2): 47–54. PubMed Abstract \| Free Full Text\n\nGosset A, Larmarange J, Mcgrath N, et al.: Retention in care trajectories of HIV-positive individuals participating in a universal test and treat programme in rural South Africa (ANRS 12249 TasP trial). J Acquir Immune Defic Syndr. 2019; 80(4): 375–385. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHendrickson CJ, Pascoe SJS, Huber AN, et al.: “My future is bright...I won’t die with the cause of AIDS”: ten-year patient ART outcomes and experiences in South Africa. J Int AIDS Soc. 2018; 21(10): e25184. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlhaj M, Amberbir A, Singogo E, et al.: Retention on antiretroviral therapy during Universal Test and Treat implementation in Zomba district, Malawi: a retrospective cohort study. J Int AIDS Soc. 2019; 22(2): e25239. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZimbawe National Statistics Agency: Zimbabwe Population Census. Harare, Zimbabwe. 2012. Reference Source\n\nMinistry of Health and Child Care and National AIDS Council: 2017 Zimbabwe HIV Estimates. Harare, Zimbabwe. 2018. Reference Source\n\nMinstry of Health and Child Care: Zimbabwe Population-based HIV Impact Assessment Report. Harare, Zimbabwe. 2016. Reference Source\n\nMinistry of Health and Child Care: Guidelines for Antiretroviral Therapy for the Prevention and Treatment of HIV in Zimbabwe. Harare, Zimbabwe. 2016; 78. Reference Source\n\nMinistry of Health and Child Care: Consolidated HIV and AIDS Job Aide. Harare, Zimbabwe. 2017. Reference Source\n\nMinistry of Health and Child Care: Guidelines for Antiretroviral Therapy for the Prevention and Treatment of HIV in Zimbabwe. Harare, Zimbabwe. 2013. Reference Source\n\nUNAIDS: 90-90-90 An ambitious treatment target to help end the AIDS epidemic. Geneva, Switzerland. 2014; 33. Reference Source\n\nRufu A, Chitimbire VTS, Nzou C, et al.: Implementation of the “Test and Treat” policy for newly diagnosed people living with HIV in Zimbabwe in 2017. Public Health Action. 2018; 8(3): 145–150. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrown LB, Getahun M, Ayieko J, et al.: Factors predictive of successful retention in care among HIV-infected men in a universal test-and-treat setting in Uganda and Kenya: A mixed methods analysis. PLoS One. 2019; 14(1): e0210126. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMagaço A, Dovel K, Cataldo F, et al.: “Good health” as a barrier and facilitator to ART initiation: a qualitative study in the era of test-and-treat in Mozambique. Cult Health Sex. 2019; 21(9): 1059–1073. PubMed Abstract \| Publisher Full Text\n\nNhassengo P, Cataldo F, Magaço A, et al.: Barriers and facilitators to the uptake of Test and Treat in Mozambique: A qualitative study on patient and provider perceptions. PLoS One. 2018; 13(12): e0205919. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nElul B, Lamb MR, Lahuerta M, et al.: A combination intervention strategy to improve linkage to and retention in HIV care following diagnosis in Mozambique: A cluster-randomized study. PLoS Med. 2017: 14(11): e1002433. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShewade HD: Matare T et study 2020 ART uptake, initiation delay and retention data. figshare. Collection. 2020. http://www.doi.org/10.6084/m9.figshare.c.4944399.v1" }	[ { "id": "62743", "date": "26 May 2020", "name": "Boniface Dongmo‐Nguimfack", "expertise": [ "Reviewer Expertise I am an economist and member of the HIV guideline Technical Working Group of the WHO" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 was designed to answer the question on uptake and retention comparing the before treat all period to the treat all period. Statistically speaking the analysis was well done and reflects the pure data analysis as well as the outcome of the analysis.\n\nThe paper lacks a clear disclaimer of the limitation(s) of the study.\n\nNo information on the lack of tracing one patient from one clinic to the other as soon as they get outside the ePMS.\n\nLack of information on the mobility of patients in the city and the country in general.\n\nLack of information on how easy or difficult it is to enroll in a new clinic.\n\nThe lack of patient unique identifier outside the ePMS clinics.\n\nThe difficulty when you reach the 80% enrolment to find the last 20%.\n\nNo comparative analysis on how easy or difficult the enrolment and the treatment was within Harare between the before treat all period to the treat all period.\n\nThe methodology does not reflect the reality of the country as there are still stigma and discrimination in the country and people tend to get tested out of their area of residence where they are not known and after enrollment tend to move closer to home for treatment which may justify the high rate of LTFU which actually may not be LTFU but enrollment in a different clinic closer to home.\n\nThe WHO not-published survey in Harare in 2018 shows that out of 100 LTFU only 25 were LTFU, 75 have just changed clinics or regions.\n\nIt is quite easy in Harare to move from one clinic to another clinic (the reason why the duplication in number is so high).\n\nWHO recommends index tracing and testing to move from 70% to 95% which is the new target.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5772", "date": "20 Aug 2020", "name": "Hemant Deepak Shewade", "role": "Author Response", "response": "Thank you very much for your comments. This has helped us improve our manuscript. We hope the revised manuscript now meets your expectations. Point by point response to your comments is provided belowReviewer:The study was designed to answer the question on uptake and retention comparing the before treat all period to the treat all period. Statistically speaking the analysis was well done and reflects the pure data analysis as well as the outcome of the analysis.Response:Thank you very much for the constructive comment. Reviewer:The paper lacks a clear disclaimer of the limitation(s) of the study.Response:Thank you very much. The strengths and limitations have been mentioned in the second last paragraph of discussion. Reviewer:No information on the lack of tracing one patient from one clinic to the other as soon as they get outside the ePMS.Response:Thank you very much. We agree with the reviewer here. We have added this as a limitation. We did not have details about what happened to those that were transferred out. Alive and on treatment and transferred out combined were combined as “retained in care”. Patients that were transferred out were censored on their last recorded visit to ART site. Reviewer:Lack of information on the mobility of patients in the city and the country in general.Response:Thank you very much. Yes, this is a limitation and is explained in second from last paragraph of the discussion section. Reviewer:Lack of information on how easy or difficult it is to enrol in a new clinic.Response:Thank you very much. We have added the following information under the methods subheading “ART initiation before “Treat All”:If a patient officially transfers-out to another ART clinic, they are issued with a transfer-out letter for presentation at the receiving ART clinic and they maintain their ART number. In other instances, patients may unofficially transfer out to another health facility without being assigned a transfer-out letter and are therefore declared lost to follow-up at their original facility if they exceed 90 days after their last scheduled clinic or drug pick-up visit and all attempts to trace them back to care have been futile. The patients will often present at the transfer-in facility with a patient-held ART booklet which indicates their previous medical history and therefore continue receiving ART care whilst also maintaining their ART number. Alternatively, if no evidence of previous treatment is provided, they are offered an HIV test and subsequently assigned a new ART number and treated as new ART client Reviewer:The lack of patient unique identifier outside the ePMS clinics.Response:Thank you very much. We agree with the reviewer. However, we included only the ePMS sites which had electronic data for analysis. Hence, this was beyond the scope of our study. We also respond to your comment in the methods subheading “ART initiation before “Treat All”” Reviewer:The difficulty when you reach the 80% enrolment to find the last 20%.Response:Thank you very much. We agree with the reviewer here based on our programme experience. Reviewer:No comparative analysis on how easy or difficult the enrolment and the treatment was within Harare between the before treat all period to the treat all period.Response:We have added some additional information in the methods section. Prior to “Treat All” psychosocial criteria for ART initiation, included completion of prescribed counselling sessions and an assessment of adherence to CPT in the past 3 months with CPT adherence being used to assess the likelihood that the patient would adhere to ART. Whereas in the Treat All era, additional changes included an emphasis for health workers to provide adequate counselling and start ART within a week with exception of pregnant and breast-feeding women who were to be started on ART on the same day of HIV diagnosis. However, for those patients who are not ready yet to start ART, they should receive on-going counselling and support. Reviewer:The methodology does not reflect the reality of the country as there are still stigma and discrimination in the country and people tend to get tested out of their area of residence where they are not known and after enrolment tend to move closer to home for treatment which may justify the high rate of LTFU which actually may not be LTFU but enrolment in a different clinic closer to home.Response:Thank you very much. We have included this point in paragraph four of the discussion section in the revised manuscript. We have copied it below for your kind perusalHigh rates of LTFU before and during “Treat All” may not actually be LTFU but enrolment in a different clinic closer to home. Due to stigma and discrimination, people tend to get tested out of their area of residence where they are not known and after enrolment tend to move closer to home for treatment. Reviewer:The WHO not-published survey in Harare in 2018 shows that out of 100 LTFU only 25 were LTFU, 75 have just changed clinics or regions.Response:Thank you very much. This supports our revision in the fourth paragraph of discussion section. Reviewer:It is quite easy in Harare to move from one clinic to another clinic (the reason why the duplication in number is so high).Response:Thank you very much. We agree with the reviewer. Reviewer:WHO recommends index tracing and testing to move from 70% to 95% which is the new target.Response:Thank you very much. We agree with the reviewer. Index tracing and testing will help achieving the first (90) or the first (95) of the UNAIDS target. This deals with what proportion of expected HIV positive should be diagnosed. Our study dealt (objective on ART uptake) with the second 90 or 95 which states what percentage of diagnosed PLHIV should be started on ART. We hope this is fine." } ] }, { "id": "64871", "date": "16 Jul 2020", "name": "Joseph Mugisha Okello", "expertise": [ "Reviewer Expertise epidemiology", "HIV and ageing", "chronic conditions" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you very much for giving me the opportunity to review this manuscript. I hope my review comments will lead to further improvement of the manuscript.\n\nGeneral comments:\nThe manuscript addresses important research questions about changes of uptake, time to initiation and retention.\n\nAdherence to ART is an important issue in the management of patients in ART programmes. I am surprised that the authors did not look at adherence while looking at key programme indicators. Although the authors indicate how monitoring of patients using viral loads was done before and after treat all, they did not look at this as one of their study outcomes. The authors need to acknowledge this as one of the weaknesses of this study.\n\nSpecific comments:\n\nIntroduction:\nIn paragraph 2 of the introduction, the authors say that research in other countries demonstrated that after implementation of the “treat all”, people on ART had better outcomes. The authors should mention these countries and specifically mention which ART outcomes were better.\n\nThe authors need to give more details on the 14% prevalence of HIV in Zimbabwe. The references given are for 2012, 2016 and 2018. Is the current HIV prevalence in Zimbabwe still 14%? How was this national prevalence of HIV estimated in Zimbabwe?\n\nIn the introduction, the authors should mention the proportion of HIV infected people who were taking ART before the treat all policy became operational and the proportion currently when treat all policy is operational.\n\nMethods:\nCan you give more information on the 77 ART sites providing HIV diagnosis and treatment? Are these public facilities or private facilities?\n\nAre there private providers of ART in Harare? If they are there, is the information captured by District health Information systems?\n\nIs data on ART adherence captured at these ART treatment sites?\n\nWhen people on ART get lost to follow up, is there anything done to establish reasons why they got lost to follow up?\n\nDiscussion:\nYou need to acknowledge the weakness of not looking at adherence in this study. You actually seem to agree with me on this point where you say “another potential reason for the observed lower retention could be deficiencies in coverage of quality adherence counselling with likely increased work load during the “treat all era”.\n\nYou also need to recommend qualitative studies to look at why retention remained low in the treat all era in Harare compared to other studies in other countries. You rightly said you speculated.\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": "5773", "date": "20 Aug 2020", "name": "Hemant Deepak Shewade", "role": "Author Response", "response": "Thank you very much for the invaluable comments. We respond below, point by point.Reviewer:Thank you very much for giving me the opportunity to review this manuscript. I hope my review comments will lead to further improvement of the manuscript.Response:Thank you very much. Reviewer:General comments:The manuscript addresses important research questions about changes of uptake, time to initiation and retention.Response:Thank you very much. Reviewer:Adherence to ART is an important issue in the management of patients in ART programmes. I am surprised that the authors did not look at adherence while looking at key programme indicators. Although the authors indicate how monitoring of patients using viral loads was done before and after treat all, they did not look at this as one of their study outcomes. The authors need to acknowledge this as one of the weaknesses of this study.Response:Thank you very much. The before period included April-June 2015 and during this time VL had not been introduced for routine monitoring of ART response. Hence, we were not able to document the before-after effect of “Treat All” on VL. Reviewer:Specific comments:Introduction:In paragraph 2 of the introduction, the authors say that research in other countries demonstrated that after implementation of the “treat all”, people on ART had better outcomes. The authors should mention these countries and specifically mention which ART outcomes were better.Response:Thank you very much. We have revised the introduction section as per the advice by reviewer. The revised lines now read as followsStudies from southern African countries have demonstrated the health and economic benefits of “Treat All”. Research in rural South Africa and Malawi demonstrated that after implementation of “Treat All”, people on ART had better retention in care. Reviewer:The authors need to give more details on the 14% prevalence of HIV in Zimbabwe. The references given are for 2012, 2016 and 2018. Is the current HIV prevalence in Zimbabwe still 14%? How was this national prevalence of HIV estimated in Zimbabwe?Response:Thank you very much. The 2018 reference is to give the updated number of PLHIV in Zimbabwe (1.3 million). The 2016 reference is the ZIMPHIA survey (Zimbabwe Population-based HIV Impact Assessment) 2015-16 which gives the estimate for HIV prevalence among adults. The 2012 reference is the census and as it is not relevant, I have removed it from here (in the third para of introduction). The revised line now reads as followsZimbabwe has a generalized HIV epidemic, with an estimated 1.3 million PLHIV and an HIV prevalence of 14% among adults (15-49 years) as per the ZIMPHIA survey 2015-16. Reviewer:In the introduction, the authors should mention the proportion of HIV infected people who were taking ART before the treat all policy became operational and the proportion currently when treat all policy is operational.Response:Thank you very much. The trends in uptake of ART among PLHIV has been described by every quarter in the results section (see Figure 1). Hence to avoid duplication, we are not mentioning the same in the introduction. Also, comparative analysis of ART uptake is one of the objectives of the study. Hence, we are not mentioning it in the introduction. We hope this is fine. Reviewer:Methods:Can you give more information on the 77 ART sites providing HIV diagnosis and treatment? Are these public facilities or private facilities?Response:Thank you very much. We have clarified in the revised manuscript that these are public facilities. Reviewer:Are there private providers of ART in Harare? If they are there, is the information captured by District health Information systems?Response:Thank you very much. Information on private providers is not captured in DHIS. Reviewer:Is data on ART adherence captured at these ART treatment sites?Response:Thank you very much. Data on ART adherence is collected but is unreliable as this is based on self-reports and pill count. Furthermore this is only available for those patients who returned for their last scheduled visit hence will not account for the LTFU and transfer-out patients. Reviewer:When people on ART get lost to follow up, is there anything done to establish reasons why they got lost to follow up?Response:Thank you very much. We have tried to speculate the reasons for this in the revised manuscript. An unpublished WHO report suggests that only 25% of those documented as LTFUs are true LTFUs. I am copy pasting the new lines added to the discussion section regarding thisHigh rates of LTFU before and during “Treat All” may not actually be LTFU but enrolment in a different clinic closer to home. Due to stigma and discrimination, people tend to get tested out of their area of residence where they are not known and after enrolment tend to move closer to home for treatment. Furthermore we have also added to the methods section in Paragraph 4 under the subheading “ART initiation before “Treat All”” that patients are declared LTFU if they are not successfully traced back to care within 90 days from their last scheduled visit either by phone call or physical follow-ups by community health workers. Reviewer:Discussion:You need to acknowledge the weakness of not looking at adherence in this study. You actually seem to agree with me on this point where you say “another potential reason for the observed lower retention could be deficiencies in coverage of quality adherence counselling with likely increased work load during the “treat all era”.Response:Thank you very much. We agree with you. But as discussed before, data on ART adherence is collected but is unreliable as this is based on self-reports and pill count. Adherence would best be measured by VL testing which was not routinely available in the period prior to HIV treat All. Hence we were not in a position to compare the same before and during Treat All. Also there were large instances of missing data of CD4 count (mentioned as a limitation in second last paragraph of discussion) Reviewer:You also need to recommend qualitative studies to look at why retention remained low in the treat all era in Harare compared to other studies in other countries. You rightly said you speculated.Response:Thank you very much. We have included a line of qualitative systematic enquiry in discussion section.The programme should consider qualitative systematic enquiry into why the retention did not improve during “Treat All” in Harare." } ] } ]	1	https://f1000research.com/articles/9-287
https://f1000research.com/articles/9-310/v1	29 Apr 20	{ "type": "Clinical Practice Article", "title": "Appearance and management of COVID-19 laryngo-tracheitis: two case reports", "authors": [ "Charles Matthew Oliver", "Marta Campbell", "Oma Dulan", "Nick Hamilton", "Martin Birchall", "Charles Matthew Oliver", "Marta Campbell", "Oma Dulan", "Nick Hamilton" ], "abstract": "We present two cases of coronavirus disease 2019 (COVID-19)-related laryngotracheitis in good-prognosis, ventilated patients who had failed extubation. As the pandemic continues to unfold across the globe and better management of those with respiratory failure develops, this may be an increasingly common scenario. Close ENT-intensivist liaison, meticulous team preparation, early consideration of rigid endoscopy and prospective data collection and case sharing are recommended.", "keywords": [ "COVID", "SARS-CoV-19", "Intensive care", "Airway management" ], "content": "Introduction\n\nCoronavirus disease 2019 (COVID-19) infection, caused by the SARS-CoV-2 virus, is presently declared a global pandemic responsible for 571,678 reported cases and 26,494 deaths at the time of writing. Initial symptoms commonly include fever and cough with a delayed onset of progressive breathlessness1. In the largest Chinese cohort of 1099 patients, mechanical ventilation was required in 2.3%2, although figures from Lombardy in Italy show higher rates and ICU bed provision has had to double in the space of six weeks3. Pressure on intensive care systems is now so great internationally that an understanding of the processes for delayed tracheal extubation is very important. We describe a patient whose extubation and discharge were delayed due to a florid COVID-19-related laryngo-tracheitis causing upper airway obstruction.\n\n\nCase report 1\n\nA 69-year-old female, non-smoker with a background history of hypertension (controlled by amlodipine 5 mg once daily) presented to the emergency department with a three-day history of pyrexia and tachycardia. Admission chest X-ray (CXR) showed bilateral pulmonary infiltrates. On day 5 after onset of symptoms, she was transferred to the intensive care unit where she required tracheal intubation and invasive ventilation for worsening type 1 respiratory failure. An 8-mm external diameter endotracheal tube (ETT, Portex, Hythe, UK) was sited on first attempt with video-laryngoscopy, secured at 22 cm at the lips, with tip position subsequently confirmed on CXR (Figure 1A)4. Laryngoscopy view was grade 15, and no pathology was recorded.\n\n(A) Post-intubation plain chest radiograph, on ICU on day 5 post-onset of symptoms. (B) Plain chest radiograph on day 10 post-onset of symptoms.\n\nWith reducing levels of ventilatory support requirement (spontaneous effort, FiO2 0.3, pressure support (PS) 5 cm H2O, positive end expiratory pressure (PEEP) 5 cm H2O, extubation was attempted five days later (day 10), but was unsuccessful due to excessive resistance to egress of the ETT. When repeat video-laryngoscopy suggested laryngeal oedema, 6.6 mg three times daily dexamethasone was commenced. Repeat CXR demonstrated no causative pathology (Figure 1B). Two further attempts at extubation over successive days again failed, characterised by lack of audible leak after cuff deflation and almost complete immobility of the tube on reasonable traction.\n\nFollowing careful planning between clinicians and managers across two sites, on day 19 the patient was transferred to an operating theatre for laryngoscopy and bronchoscopy. Ventilatory parameters were unchanged, she required no additional organ support and only minimal sedation (propofol and fentanyl) was required to ensure ETT tolerance. On the day of surgery, two iterative team briefs were conducted, during which all team members were asked to contribute questions and suggestions; a plan was agreed with all potential anticipated events and adverse events considered, along with their mitigation, and equipment located.\n\nAll team applied full personal protective equipment (PPE), comprising: FFP3 mask (fit-checked and leak-tested by trained testers), visor, apron, gown, two pairs of gloves and PPE footwear. Communication between in-theatre staff (‘COVID-19 team’) and external support staff (nurse and ODP, non-COVID team) was established with two-way radios. The patient was transferred onto an anaesthetic ventilator, neuromuscular blockade administered (rocuronium 50 mg) and ventilated on mandatory mode, FiO2 1.0. General anaesthesia was aintained with propofol and fentanyl infusions and further rocuronium boluses were administered. A tracheostomy set was prepared with size 6 and 7 cuffed non-fenestrated tubes (Portex, Hythe, UK) tested and pre-loaded with introducers in case of upper airway obstruction.\n\nLaryngoscopy was performed using a combination of adult Lindholm and Dedo laryngoscopes (Karl Storz, Jena, Germany), to visualise the supraglottis and glottis respectively. Laryngoscopes were placed on suspension without the need for counter-pressure and imaging performed using a 0o Hopkins’ rod telescope and camera system (Karl Storz, Jena, Germany). Bronchoscopy via a T-piece port attached to the ETT was performed using a disposable bronchoscope (Broncho Slim, Ambu, Ballerup, Denmark). This showed that the lower trachea, main and lobar bronchi were normal with no obvious mucosal oedema, excessive secretions or ulceration.\n\nThe epiglottis was inflamed with shallow, irregular, ulcers (Figure 2A). A sample of the ulcerated area was sent for microbiology testing. The rest of the supraglottis and superior surface of the vocal cords were spared, whilst profound oedema encased the ETT from cord level downwards (Figure 2B). It was not possible to pass the Hopkins’ rod past cord level. Adrenaline 1:10,000-soaked neurosurgical patties were packed around the tube in the glottic and subglottic area for 15 minutes to try and reduce swelling and risk of bleeding, and then removed using microlaryngeal instruments. Following pre-oxygenation and apnoea, a paediatric endotracheal tube bougie (10 ch × 600 mm, P3 Medical Ltd) was introduced through the ETT, the ETT was removed atraumatically with steady traction and a size 6 ETT then “railroaded” over the bougie (under direct rigid laryngoscopic) vision to replace it. Ventilation was recommenced without incident. Hopkins’ rod examination was now possible through the newly patent anterior glottis (Figure 2C), but only as far as the fourth tracheal ring due to upper tracheal and subglottic oedema. Ulcers were present bilaterally in the subglottis (Figure 2D). Depomedrone (40 mg/ml, 0.3 ml per side) was injected into the subglottis using a modified butterfly needle.\n\n(A) View of supraglottis showing ulcerated epiglottis. (B) Glottis showing relative sparing of vocal cords and false cords, but profound subglottic oedema. (C) Following change to size 6 endotracheal tube, there is some anterior glottic airway. (D) However, the subglottis is also ulcerated and oedematous mucosa prevents rigid bronchoscopy (0o Hopkins’ rod) beyond the third tracheal ring. White arrows indicate areas of ulceration and red arrow subglottic oedema.\n\nThe theatre team “doffed” (removed protective clothing) in a dedicated anteroom, immediately adjacent to the operating theatre and showered. A debrief was then held where all learnings, thoughts and feelings were recorded. The values of planning, repetition of plans, risk anticipation and effective communication and egalitarian team-work were highlighted. Problems identified were the difficulties in communicating verbally between theatre staff and between those inside and outside theatre due to protective clothing and protocols, and the time and expertise required to prepare adequately and safely for a high-risk COVID-19 airway case.\n\nThe patient was returned to ICU following the procedure, where supportive treatment and systemic corticosteroid treatment was continued. On day 23, following confirmation of ‘cuff leak’, she was successfully extubated. On day 25 she was stepped down to a level 1 bed.\n\n\nCase report 2\n\nA 45-year-old female with poorly controlled, insulin-dependent diabetes mellitus (with retinopathy), hypothyroidism and central adiposity presented to our emergency department in extremis, in diabetic ketoacidosis, severely dehydrated and agitated following two days of cough and anorexia. The cough was non-productive. Arterial blood gas results included pH 6.91, Base -26, blood sugar level was high (unrecordable) and ketones were elevated at 5 mmol/L. A size 7.0-mm cuffed oral endotracheal tube was chosen to permit invasive ventilation and bronchoscopy if required; Cormac & Lehane view was Grade 2 and the tube was fixed at 23 cm from the lips. Initial CXR, Figure 3a. Medications on presentation were metformin 1 g twice daily, Lantus & Novorapid (variable doses) and levothyroxine 100 µg once daily.\n\n(A) Post-intubation plain chest radiograph, on day 1 of hospital admission. (B) Day 5 following re-intubation.\n\nShe was transferred to an isolation room on the main intensive care unit, started on a fixed rate intravenous insulin infusion (0.1 units/kg/h), fluid resuscitated and started on ceftriaxone (per protocol) and clarithromycin.\n\nOn day 5 of admission, the ETT was removed in a trial of extubation. She was stridulous, not improving with nebulised adrenaline and intravenous corticosteroids, and progressively developed increased work of breathing. She was re-intubated (again size 7) several hours later and started on regular dexamethasone 6.6mg TDS. Subsequent CXR, Figure 3b.\n\nOn day 13 she remained suitable for extubation by pulmonary and other measures, but no cuff leak was present when assessed. On day 15 she underwent a surgical tracheostomy preceded by microlaryngoscopy and bronchoscopy. At microlaryngoscopy there was profound oedema in the glottis and subglottis (Figure 4). Passage of a disposable fine-bore bronchoscope (Broncho Slim, Ambu, Ballerup, Denmark) through the anterior commissure revealed extensive tracheal oedema with some granulation tissue and ulceration in the subglottis. It was deemed impossible to extubate due to the swelling and so tracheostomy was performed according to the UCLH COVID19 tracheostomy protocol. In brief, through a small collar incision, the trachea was approached using only clips and ties to reduce the risk of inhaled virus-rich “plume” from diathermy. After pre-oxygenation, the ETT was advanced beyond the site of the tracheostomy with the balloon fully inflated and ventilation suspended. A window was created revealing again oedematous mucosa and the endotracheal tube withdrawn under direct vision until the tip was just higher than the window. A size 7 tracheostomy tube (Blueline Ultra, PORTEX, Hythe, Kent) was placed. A pre-loaded closed suction and ventilation extension, with a viral filter, was attached, the cuff inflated, and ventilation recommenced. The tube was sewn in place at all four poles and ties added. Post-operatively she steadily improved and, on day 22, tracheostomy wean was progressing well. By day 7 after surgery, intraoperative samples had grown no pathological bacteria.\n\n(A) View of supraglottis showing ulcerated glottis. (B) Glottis showing sparing of false cords, but profound glottic oedema and glottic and subglottic ulceration. (C) Flexible bronchoscopy via the anterior commissure shows subglottic oedema and granulation tissue (black arrow). (D) Oedematous mucosa prevents flexible bronchoscopy beyond the third tracheal ring. White arrows indicate areas of ulceration, black arrow granulation tissue and red arrow tracheal oedema.\n\n\nDiscussion\n\nViral upper airway infection may present as a spectrum ranging from dysphonia to fulminant airway compromise, representing oedema, inflammation and ulceration. In a literature review, we identified case reports of clinically significant epiglottitis, laryngitis and tracheitis associated with less commonly encountered viral pathogens (HSV, HZV and HIV)6–8. Anecdotally, glottic oedema has been seen as a presenting feature of COVID-19 in an infant (C. Frauenfelder, Great Ormond Street Hospital for Children, personal communication 29th March 2020). However, upper airway involvement has however yet to be formally reported in coronavirus infection in humans to our knowledge.\n\nThe coronavirus enters cells by binding to the angiotensin converting enzyme 2 (ACE2) receptor which is found on the apical surface of differentiated ciliated respiratory epithelia9–11. This cell type is particularly dense in airway epithelial cells, hence the severity of COVID-19 disease in lungs and distal airways. However, the adult glottic and supraglottic larynx has variable areas of ciliated respiratory cells12, which may explain why only parts of the supraglottis were affected whilst the subglottis and trachea were profoundly oedematous. In chickens, coronavirus infection is associated with laryngotracheitis13, but this condition has not previously been described in primates or humans.\n\nThese cases highlight the need for close interdisciplinary working and communication in the management of airway complications of COVID-19 infection. Here, careful joint planning between anaesthetists and ENT (laryngology specialist) surgeons was critical. We recommend daily laryngology/head and neck surgeon meetings with ICU staff during such pandemics ideally through the use of video conferencing software to limit potential spread between healthcare workers. Meetings should discuss issues on a case-by-case basis with written protocols designed to carefully balance risk and benefit of, especially, tracheostomy. In the first case presented, such dialogue obviated the need for tracheostomy.\n\nFull PPE and COVID-19 protocols require a new approach to theatre communication. Task-specific equipment, such as disposable ear-pieces or throat microphones, might be developed where they do not compromise mask seals. Communication protocols, such as those used by airlines and the military, may be introduced.\n\nThe key findings in the present cases were ulceration of the epiglottis and subglottis and profound oedema and granulations in the subglottis and upper trachea. These changes were observed despite resolution of clinical, radiological and bronchoscopic characteristics of COVID-19 respiratory disease and clinical improvement based on reduction in oxygen and ventilation needs. The relatively late and prolonged response of this part of the airway may be idiosyncratic and the true incidence and demographics of COVID-19 laryngotracheitis (C19LT) will only be understood by prospective national/multinational case and data collection.\n\nPrior to the theatre procedure, we used systemic steroids to try and reduce upper airway oedema. In the present cases, its use did not avoid the ultimate need to resort to rigid endoscopy and experience with previous SARS epidemics suggest systemic steroids may increase viral shedding14. We hypothesise that early consideration of such endoscopy, especially in “good prognosis” patients, may be indicated rather than a trial of steroids. Likewise, it could be argued that an intra-laryngeal injection of depot steroids in the first case may slow rather than assist local resolution of oedema. Again, prospective data collection is required to answer these questions.\n\nTracheostomy represents the third highest risk of COVID-19 transmission to staff after ETT intubation and non-invasive ventilation15. Reports from Hong Kong, which experienced high levels of SARS-1 and SARS-2 cases, highlights the need to delay or avoid tracheostomies in this group of patients where clinically possible16–18. Whether tracheostomy can expedite extubation and free up ventilator capacity during the COVID-19 pandemic is not yet established and should be the focus of research activity. The narrowing, oedema and ulceration of the trachea in exactly the location where a tracheostomy, either open or percutaneous, would be performed suggests that such procedures may be more hazardous and present more post-operative problems than in those without such oedema. In selected cases, rigid endoscopy may be useful in defining the pathology.\n\nCoronavirus may cause symptomatic inflammation of the larynx as well as the trachea, bronchi and lungs, resulting in difficulties in both tracheal intubation and extubation.\n\nA distinct condition of COVID-19-related laryngotracheitis may exist. This may make siting of tracheostomy tubes even more problematic due to narrowing of the airway, thickening of mucosa and increase in local secretions.\n\nEarly consideration of this diagnosis and endoscopy may be considered.\n\nTracheal intubation and extubation of the patient with COVID-19 may be a high-risk procedure for staff, irrespective of the clinical severity of disease. Where possible, Aerosol generating procedures (AGP) should be performed in a negative pressure room with > 12 air changes per hour whenever possible.\n\nTracheal intubation and extubation of the patient with COVID-19 may be a high-risk procedure for staff, irrespective of the clinical severity of disease.\n\nMeticulous planning with the full theatre team is required before embarking on all airway procedures in COVID19 infected patients.\n\nCommunication issues due to the wearing of PPE in operating theatres require novel solutions.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patients.", "appendix": "References\n\nHuang C, Wang Y, Li X, et al.: Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020; 395(10223): 497–506. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuan WJ, Ni ZY, Hu Y, et al.: Clinical Characteristics of Coronavirus Disease 2019 in China. N Engl J Med. 2020; NEJMoa2002032. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDavenport L: ICU Lessons on COVID-19 From Italian Front Line: Be Flexible. 2020; (accessed 29.03.20 2020). Reference Source\n\nAnaesthetists RCo: Royal College of Anaesthetists COVID-19 clinical guidance. 2020; (accessed 29.03.20 2020). Reference Source\n\nCormack RS, Lehane J: Difficult tracheal intubation in obstetrics. Anaesthesia. 1984; 39(11): 1105–11. PubMed Abstract \| Publisher Full Text\n\nHarless L, Jiang N, Schneider F, et al.: Herpes Simplex Virus Laryngitis Presenting as Airway Obstruction: A Case Report and Literature Review. Ann Otol Rhinol Laryngol. 2017; 126(5): 424–8. PubMed Abstract \| Publisher Full Text\n\nDominguez LM, Simpson CB: Viral laryngitis: a mimic and a monster - range, presentation, management. Curr Opin Otolaryngol Head Neck Surg. 2015; 23(6): 454–8. PubMed Abstract \| Publisher Full Text\n\nTebruegge M, Connell T, Kong K, et al.: Necrotizing Epiglottitis in an Infant: An Unusual First Presentation of Human Immunodeficiency Virus Infection. Pediatr Infect Dis J. 2009; 28(2): 164–6. PubMed Abstract \| Publisher Full Text\n\nJia HP, Look DC, Shi L, et al.: ACE2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia. J Virol. 2005; 79(23): 14614–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRen X, Glende J, Al-Falah M, et al.: Analysis of ACE2 in polarized epithelial cells: surface expression and function as receptor for severe acute respiratory syndrome-associated coronavirus. J Gen Virol. 2006; 87(Pt 6): 1691–5. PubMed Abstract \| Publisher Full Text\n\nTo KF, Lo AW: Exploring the pathogenesis of severe acute respiratory syndrome (SARS): the tissue distribution of the coronavirus (SARS-CoV) and its putative receptor, angiotensin-converting enzyme 2 (ACE2). J Pathol. 2004; 203(3): 740–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStell PM, Gregory I, Watt J: Morphometry of the epithelial lining of the human larynx. I. The glottis. Clin Otolaryngol Allied Sci. 1978; 3(1): 13–20. PubMed Abstract \| Publisher Full Text\n\nNakamura K, Imai K, Tanimura N: Comparison of the effects of infectious bronchitis and infectious laryngotracheitis on the chicken respiratory tract. J Comp Pathol. 1996; 114(1): 11–21. PubMed Abstract \| Publisher Full Text\n\nLee N, Allen Chan KC, Hui DS, et al.: Effects of early corticosteroid treatment on plasma SARS-associated Coronavirus RNA concentrations in adult patients. J Clin Virol. 2004; 31(4): 304–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTran K, Cimon K, Severn M, et al.: Aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review. PLoS One. 2012; 7(4): e35797. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAhmed N, Hare GM, Merkley J, et al.: Open tracheostomy in a suspect severe acute respiratory syndrome (SARS) patient: brief technical communication. Can J Surg. 2005; 48(1): 68–71. PubMed Abstract \| Free Full Text\n\nHo OY, Lam HC, Woo JK, et al.: Tracheostomy during SARS. J Otolaryngol. 2004; 33: 393–6. PubMed Abstract \| Publisher Full Text\n\nMorgan P: Tracheostomy in a patient with SARS. Br J Anaesth. 2004; 92: 905–6; author reply 6. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "63333", "date": "15 May 2020", "name": "Jeyasakthy Saniasiaya", "expertise": [ "Reviewer Expertise Otorhinolaryngology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 a clear, well-written manuscript. Adequate information especially on patient's presentation as well as approach was described. The introduction is relevant. Adequate information on previous study findings on viral laryngotracheitis is mentioned for readers to follow. Also, it is interesting that step-by-step airway assessment was provided for readers which is especially prudent during this period. However, the authors need to mention on why biopsy was not performed in this case. This manuscript definitely adds value to the current COVID-19 pandemic.\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? 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 conclusion balanced and justified on the basis of the findings? Yes", "responses": [ { "c_id": "5799", "date": "20 Aug 2020", "name": "Charles Matthew (Matt) Oliver", "role": "Author Response", "response": "Thank you for your reviews, which we have used to improve the quality of this manuscript.Regarding biopsies: We wanted to reduce the potential for transmission as much as possible. In retrospect, we would have taken a biopsy if we knew what we know now and had updated systems in place." } ] }, { "id": "65373", "date": "15 Jul 2020", "name": "Jean Paul Marie", "expertise": [ "Reviewer Expertise I am an ENT", "specialized in airway and neurosciences" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 : \"We describe a patient whose extubation and discharge were delayed due to a florid COVID-19-related laryngo-tracheitis causing upper airway obstruction.\" The title of the paper is now 2 patients. Has to be corrected.\n\nCase report 1 “An 8-mm external diameter endotracheal tube (ETT, Portex, Hythe, UK)\" as it a n° 8 portex tube. Are you sure that external diameter is 8 mm?\n\nCase report 2 OK.\n\nDiscussion Well built.\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? 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 conclusion balanced and justified on the basis of the findings? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-310
https://f1000research.com/articles/8-859/v1	13 Jun 19	{ "type": "Systematic Review", "title": "Humanistic burden and economic impact of heart failure – a systematic review of the literature", "authors": [ "Lucia Giles", "Caroline Freeman", "Polly Field", "Elisabeth Sörstadius", "Bernt Kartman", "Lucia Giles", "Caroline Freeman", "Polly Field", "Elisabeth Sörstadius" ], "abstract": "Background: Heart failure (HF) is increasing in prevalence worldwide. This systematic review was conducted to inform understanding of its humanistic and economic burden. Methods: Electronic databases (Embase, MEDLINE®, and Cochrane Library) were searched in May 2017. Data were extracted from studies reporting health-related quality of life (HRQoL) in 200 patients or more (published 2007–2017), or costs and resource use in 100 patients or more (published 2012–2017). Relevant HRQoL studies were those that used the 12- or 36-item Short-Form Health Surveys, EuroQol Group 5-dimensions measure of health status, Minnesota Living with Heart Failure Questionnaire or Kansas City Cardiomyopathy Questionnaire. Results: In total, 124 studies were identified: 54 for HRQoL and 71 for costs and resource use (Europe: 25/15; North America: 24/50; rest of world/multinational: 5/6). Overall, individuals with HF reported worse HRQoL than the general population and patients with other chronic diseases. Some evidence identified supports a correlation between increasing disease severity and worse HRQoL. Patients with HF incurred higher costs and resource use than the general population and patients with other chronic conditions. Inpatient care and hospitalizations were identified as major cost drivers in HF. Conclusions: Our findings indicate that patients with HF experience worse HRQoL and incur higher costs than individuals without HF or patients with other chronic diseases. Early treatment of HF and careful disease management to slow progression and to limit the requirement for hospital admission are likely to reduce both the humanistic burden and economic impact of HF.", "keywords": [ "heart failure", "health-related quality of life", "systematic literature review", "humanistic burden", "economic burden", "healthcare cost", "hospitalization", "readmission rates" ], "content": "Introduction\n\nHeart failure (HF) is estimated to affect more than 23 million people worldwide1 and is increasing in prevalence2. Causes of HF include those of cardiac aetiology in addition to chronic diseases such as diabetes mellitus (DM) and chronic kidney disease (CKD)3; HF has been reported as the most common cardiovascular complication of DM4. The growing prevalence of HF can be attributed both to the increasing median age of populations worldwide and to the increasing prevalence of cardiovascular disease, obesity, and DM1,4. Early treatment of DM could have a significant impact in preventing the development of HF in a proportion of patients, limiting both the humanistic burden – the effect on the health-related quality of life (HRQoL) of the individuals affected5 – and the economic impact of the disease.\n\nHF is chronic and progressive in nature, and is classified into New York Heart Association (NYHA) classes I–IV based on physical limitation and objective assessment of the presence of cardiovascular disease6. The signs and symptoms of HF include breathlessness, swelling, fatigue, and fluid retention, which can lead to reduced mobility and impaired daily physical functioning7. Patients may also face psychological problems, such as depression or anxiety, as well as social concerns, especially regarding isolation8. The impact of HF symptoms upon multiple aspects of patients’ lives means that the condition can cause a significant reduction in HRQoL9. Both HF-specific and generic HRQoL instruments can be used to measure the effects of HF on patients’ daily lives and well-being10.\n\nMost patients with HF require routine management of their disease9. Treatment typically comprises daily medication as well as periodic visits to primary care providers; however, the most economically costly aspect of the disease is admission to hospital and subsequent inpatient care11. This is often required owing to worsening of symptoms. The acute nature of these occurrences, along with the risk of infection, can mean that patients with HF may be admitted to hospital as an emergency case12, incurring high costs13. Patients are typically treated in the intensive care unit, with clinical stabilization and symptom improvement as the primary focus following admission12. Once a patient’s condition has stabilized, they are likely to be transferred to the ward, and a multidisciplinary approach employed to aid disease management12. Treatment optimization and the management of both cardiovascular and non-cardiovascular comorbidities play a significant role in preventing further hospitalizations, because hospitalization with HF is associated with high rates of readmission12. Outside hospital, many patients with HF receive care in a primary setting or may receive informal care and support in the home, imposing a wider societal cost14.\n\nThis systematic review (SR) was conducted to inform understanding of the humanistic burden and economic impact of HF by identifying relevant evidence on HRQoL, costs, and medical resource use in patients with HF.\n\n\nMethods\n\nA systematic search was performed using MEDLINE® and MEDLINE In-Process, Embase, and the Cochrane Library via Ovid between January 2002 and May 2017 for the humanistic burden SR, and between January 2007 and May 2017 for the economic impact SR. Supplementary searches included reviews of congress abstracts between 2015 and 2017 (or the most recent 2 years available) for the following meetings: International Society for Pharmacoeconomics and Outcomes Research US and European congresses, American Heart Association Scientific Sessions, European Society of Cardiology (ESC) Congress, World Congress of Cardiology and Cardiovascular Health, American College of Cardiology Annual Scientific Session, and ESC Heart Failure. The search strings used to identify evidence are listed in Table 1.\n\nAbstracts and titles identified were screened by an independent reviewer to determine whether they met the PICOS (patient, interventions, comparisons, outcomes, and study design) eligibility criteria (Table 2), in accordance with 2009 Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines15. All publications that met the entry criteria for the review were obtained as full articles and reassessed against the review criteria. Owing to the large number of citations meeting the predefined inclusion criteria, a decision was made to restrict data extraction from eligible studies. For both SRs, data were extracted only from full publications of observational studies. Data were not extracted if the study population included fewer than 200 patients for the humanistic burden SR, and fewer than 100 patients for the economic impact SR. The restriction criteria used are summarized Table 2.\n\nHF, heart failure. HRQoL, health-related quality of life. NYHA, New York Heart Association. SR, systematic review.\n\nFor each publication, information was extracted into a data-extraction table. For each study, NYHA classification, age, and comorbidities including CKD and DM were recorded.\n\n\nResults\n\nIn the initial searches, 11 622 papers were identified, of which 2186 were removed as duplicates, and 9436 papers were included for electronic screening. Electronic screening identified 8166 papers that did not meet the inclusion criteria. In total, 124 papers were identified for inclusion following full paper review: 54 papers reporting HRQoL and 71 papers reporting costs and resource use (including one reference that reported data for both outcomes). A PRISMA flow diagram is shown in Figure 1. The studies identified were grouped according to the key data presented, as shown in Figure 2.\n\nHRQoL, health-related quality of life. PRISMA, Preferred Reporting Items for Systematic Reviews and Meta-Analyses.\n\nHF, heart failure. HRQoL, health-related quality of life. aSome studies reported data that fell into more than one category.\n\nA small number of studies assessed HRQoL in patients with HF compared with individuals without HF, or patients with other chronic diseases. In a study conducted in the Netherlands, patients with HF (NYHA class II–IV) reported worse scores across all domains of the 36-item Short-Form Health Survey (SF-36) than a population of age- and sex-matched community control individuals (Figure 3)16. A Danish study also found that patients who had been discharged from hospital reported lower 12-item Short-Form Health Survey (SF-12) physical component summary (PCS; mean±standard deviation [SD]: 42.5±11.1 vs 47.7±13.4, p<0.001) and mental component summary (MCS; mean±SD: 48.2±10.9 vs 51.5±14.7, p<0.001) scores than a reference group representative of the national population17. A further cohort survey, conducted in the UK, compared patients with HF with those who had asthma, chronic obstructive pulmonary disease (COPD), DM, epilepsy, or stroke. Patients with HF reported lower baseline 5-dimension EuroQol questionnaire (EQ-5D) utility and visual analogue scale (VAS) scores than patients with asthma, DM, epilepsy, and stroke, and similar scores to patients with COPD18.\n\nThis figure provides a graphical representation of the component scores for the SF-36 HRQoL questionnaire for patients with HF (stage II–IV) compared with community-dwelling elderly people who do not have HF. BP, bodily pain. GHP, general health perception. HF, heart failure. HRQoL, health-related quality of life. MH, mental health. PF, physical functioning. RL-M, role-limiting (mental). RL-P, role-limiting (physical). SF, social functioning. SF-36, 36-item Short-Form Health Survey. V, vitality.\n\nStudies from Sweden19, Australia20, and the USA21,22 assessed HRQoL by NYHA HF class and reported a greater humanistic burden with more severe disease (Figure 4). Based on the evidence identified, there was a reduction in HRQoL between NYHA HF classes I and II19,21,22 and classes II and III19–22, and between classes III and IV19–22, assessed using the Minnesota Living with Heart Failure Questionnaire (MLHFQ) and the Kansas City Cardiomyopathy Questionnaire (KCCQ), both of which are cardiac-specific instruments. One study also reported decreasing SF-36 PCS and MCS scores with increasing NYHA HF class19. For SF-12 scores, there was a greater reduction between NYHA classes I and II, and smaller reductions between more severe NYHA classes (Figure 4). A consistent decrease in utilities between classes II and III (–0.042) and classes III and IV (–0.041), measured using the 5-level EQ-5D (EQ-5D-5L) was also reported by a single study alongside a decline in MLHFQ scores (Figure 4)20.\n\nThis figure provides a graphical representation of HRQoL scores by NYHA class as measured by the MLHFQ, KCCQ, SF-36, or EQ-5D-5L questionnaires. Better health-related quality of life is represented by higher KCCQ, SF-36, and EQ-5D-5L scores, and lower MLHFQ scores. EQ-5D-5L, 5-level, 5-dimension EuroQol questionnaire. HFpEF, heart failure with preserved ejection fraction. HFrEF, heart failure with reduced ejection fraction. HRQoL, health-related quality of life. KCCQ, Kansas City Cardiomyopathy Questionnaire. MCS, mental component summary. MLHFQ, Minnesota Living with Heart Failure Questionnaire. NYHA, New York Heart Association. PCS, physical component summary. SF-36, 36-item Short-Form Health Survey.\n\nTwo further studies, both conducted in the USA, reported a negative correlation between HRQoL and increasing HF severity. A study of patients with HF living in a rural location reported a negative association between NYHA HF class and MLHFQ score, but did not report results by separate NYHA classes23. Another study investigated HRQoL in a subgroup of patients with HF as a comorbidity, from a population with CKD in a subanalysis of the Hemodialysis Study, a randomized controlled trial of patients undergoing haemodialysis. Reductions in both mental and physical HRQoL were reported with increasing HF severity (categories ranging from ‘absent’ to ‘severe’). Scores in the role-limiting (physical), bodily pain, general health, vitality, social functioning, and mental health domains, as well as overall PCS scores, decreased with increasing HF severity24.\n\nFew longitudinal studies reporting HRQoL in HF were identified. Of the studies that were purely observational, two reported a decrease in HRQoL over time, whereas the remaining three reported an improvement in HRQoL across the study period. A UK cohort study investigating HRQoL in patients with HF relative to those with other chronic diseases reported generic and disease-specific HRQoL scores at baseline and 1 year. Patients with HF were the only group to report a reduction in HRQoL over the follow-up period, with significantly lower EQ-5D VAS scores indicating worse HRQoL at 1 year18. A US study reported HRQoL over a follow-up of up to 6.6 years, adjusting for the effect of time on disease deterioration24. PCS scores decreased over time, along with scores for six of the eight SF-36 domains; MCS scores and scores for the role-limiting (mental) domain did not decrease over time24.\n\nA study in the Netherlands, however, reported that patients with a higher level of education experienced a statistically significant improvement in the role-limiting (emotional) domain of the SF-36 across an 18-month longitudinal study (p=0.015). No significant difference was found in the remaining SF-36 domains25. Patients in a Canadian study in whom HF was diagnosed following an emergency department (ED) visit reported increases in physical HRQoL scores over a 12-month period (SF-12 PCS score: 1.6–3.4; KCCQ-physical limitation score: 5.6–9.4)26. An analysis modelled health trajectories over 12 months defined by patients’ KCCQ scores over time using data from the Patient-Centered Disease Management for Heart Failure trial. Patients with a poor or moderate health status trajectory experienced improvements in KCCQ scores between baseline and 3 months followed by a stabilization in scores up to 12 months, whereas those who had a trajectory of ‘marked improvement’ reported an increase in HRQoL over the whole 12-month period27.\n\nIn a number of the longitudinal studies identified, healthcare professionals provided support to patients over the follow-up period, which was considered likely to impact HRQoL. These studies are summarized in Extended data: Supplemental Table 128. Studies in Norway29 and in Spain30 reported a statistically significant improvement in MLHFQ scores between baseline and 6 or 12 months, respectively, for patients monitored over the study period. In the Spanish study, patients continued to experience an increase in HRQoL over 5 years30. An improvement in MLHFQ scores over 12 months following enrolment into an HF clinic was also reported by patients in a Canadian study31.\n\nFew studies comparing HRQoL in patients with HF with preserved ejection fraction (HFpEF) and those with HF with reduced ejection fraction (HFrEF) were identified. In a study conducted in Germany, patients with HFpEF reported better physical functioning, measured using the SF-36, than patients with HFrEF32. A study conducted in the USA reported that patients with HFpEF experienced no greater impact on HRQoL than those with HFrEF as disease severity increased. However, patients with HFpEF, with KCCQ scores in the lowest quartile, had a higher risk of mortality or hospitalization as well as lower rates of event-free survival than those who had scores in the highest quartile21.\n\nComorbidities in patients with HF were typically associated with reductions in HRQoL (Extended data: Supplemental Table 128). In studies conducted in Spain and the USA, respectively, patients with HF and iron deficiency33 or pain34 reported higher overall and physical summary MLHFQ scores, representative of worse HRQoL, than those without these comorbidities. However, a further study in the USA found that although patients with anaemia reported worse baseline KCCQ scores than those without, deterioration in HRQoL over a 3-month period was similar in both populations22. Patients in a study in the Netherlands who experienced difficulties with sexual activity reported worse HRQoL than those without difficulties35. In a US study, excessive daytime sleepiness and cognitive impairment were associated with comparatively lower HRQoL36. In a second US study, patients who had comparatively lower haemoglobin levels at baseline reported worse KCCQ and MLHFQ scores than those with higher haemoglobin levels37.\n\nPatients in Australia, the UK, Ireland, the USA, and Norway who had ischaemic heart disease with ischaemic HF, angina, or a myocardial infarction as a comorbidity reported PCS scores that were lower than expected for the general population (more than one SD below the standardized mean of the general US population), whereas MCS scores were within one SD of the mean38.\n\nPoor mental health was generally associated with worse HRQoL in patients with HF. Comparative studies were identified that focused on depression, suicidal ideation, panic disorder, and distress (Extended data: Supplemental Table 128).\n\nPatients with HF and depression had worse HRQoL than those without depression in studies from Germany, Italy, and the USA. Statistically significant reductions in scores across all SF-36 domains and the KCCQ summary domains were found with increasing severity of depression in two studies in Germany39,40. A further German study reported an association between lower SF-36 physical functioning (PF) and bodily pain scores as severity of depression increased41. Furthermore, in a study in the USA, PF was negatively correlated with depression42. Worse health status, as defined by KCCQ score, was associated with a comparatively higher risk of depression at baseline in another US study27; and a German study indicated that patients with type D personality, predictive of depression, reported worse HRQoL (MLHFQ scores) than those with a different personality type43.\n\nSuicidal ideation and ideas of self-harm were reported to be associated with reduced HRQoL44. Patients with panic disorder45 and those who experienced distress at baseline and/or follow-up46 reported worse HRQoL than those who did not. Another study reported an association between patients’ end-of-life preferences and their physical HRQoL (SF-36 PCS scores)47.\n\nPatients’ perceived health status was reported to have a moderate impact on MLHFQ score in a Canadian study48, and social functioning, measured using the KCCQ, was found to be a predictor of perceived health status, particularly in male patients with HF in the USA49. High levels of self-care were associated with better MLHFQ or KCCQ scores in studies in Italy50 and the USA51, respectively, as well as lower hospitalization rates in the Italian study.\n\nPatients hospitalized for HF experienced comparatively better HRQoL once discharged from hospital (Extended data: Supplemental Table 128). A study in the USA and Canada found that EQ-5D scores were higher at the time of hospital discharge than when patients were admitted to hospital (baseline), and remained higher than baseline 30 days after discharge from hospital52. Another US study showed that MLHFQ scores improved significantly during the first month post-discharge (p<0.001)53. Furthermore, patients in a study in the USA who did not experience readmission to hospital within 30 days of discharge experienced better HRQoL (KCCQ scores) than those who did54. Fear of hospitalization had a negative impact on patients’ MLHFQ scores in an Italian study55, and unplanned hospitalization was identified as a predictor of better SF-12 PCS scores in a patient population who had previously been hospitalized for HF in a study in Denmark17.\n\nOwing to the chronic nature of HF, the implementation of appropriate disease management is an important part of treatment, and evidence identified in this SR indicates that it is associated with HRQoL improvements (Extended data: Supplemental Table 128). Patients in the UK56 and Germany57 with HF and either atrial fibrillation or sinus rhythm reported an improvement in HRQoL following cardiac resynchronization therapy and treatment with β-blockers, respectively. The use of left ventricular assist devices (LVADs) as bridging or destination therapy was also associated with an improvement in HRQoL in a US study58, and patients with an LVAD had better HRQoL than patients assessed for, or awaiting, heart transplant in a UK study59.\n\nSeveral studies were identified that reported HRQoL as a predictive factor both of mortality and of outcomes in patients with HF (Extended data: Supplemental Table 128). Scores in the SF-36 and SF-12 general health and PF domains were predictors of mortality in studies conducted in the Netherlands60 and the USA61, and were associated with an increased risk of hospitalization and ED visits in another study in the USA62. Two further studies, one in Italy63 and the other multinational64, found that KCCQ score was a predictor of mortality, and a study in the USA reported a negative association between MLHFQ score and cardiac event-free survival65. Physical and depressive symptoms, in addition to spiritual well-being and comorbidity count, were negatively correlated with HF-specific health status (partially determined by KCCQ score) in patients in the USA66. An Australian study examined the contribution of MLHFQ and EQ-5D scores to composite scoring systems in HF trials67.\n\nIn several studies, patient ethnicity and sex were shown to impact HRQoL (Extended data: Supplemental Table 128). A study in the USA found that Hispanic patients reported better HRQoL than those of white or black ethnicity68. In two Canadian studies, women reported worse physical and overall HRQoL, respectively, than men at baseline26,69, and worse physical HRQoL at 12 months69. Sociodemographic factors including marital status, education, income, and employment were correlated with worse HRQoL in studies in the Netherlands25 and in Canada, France, and the USA70.\n\nThe overall costs associated with the treatment for HF varied widely across studies. This is likely to result from disparities in study settings and populations, which meant that the patients included differed in terms of disease severity, clinical history, and comorbidities.\n\nFive studies conducted in Europe reported total annual healthcare costs associated with HF. The lowest annual per-patient costs reported were €3150 in a German study71, followed by €5700 in a Swedish study72, and €6571 in a study conducted in Spain73. Two Italian studies, each of which included only patients who had experienced an HF-related hospitalization, reported total annual costs of €11 10074 and €11 86475, respectively.\n\nThe lowest total annual costs reported in the USA were for a subpopulation of individuals enrolled in Medicare; patients with HF who were not classed as having a low income or dual eligibility for Medicare and Medicaid (non-low-income/dually eligible [LI/DE] cohort) incurred per-patient costs of US$13 897 annually. This value was US$17 840 for patients with HF who were classed as LI/DE76. The highest annual US costs identified were from a study comparing patients with HF who died within 1 year and those who survived. Surviving patients incurred total mean per-patient costs of US$36 426 per annum77. A further four US studies reported annual per-patient costs in the range of US$16 912–29 45678–81. Two additional US studies recorded total costs for patients in the period before they died as a result of HF; these data are not included in the overall range, but are presented in Extended data: Supplemental Table 282–84. In addition to evidence from Europe and the USA, total annual costs associated with HF were reported as C$27 809 in a Canadian study85 and ¥28 974 in a study from China86.\n\nRather than reporting annual total costs for HF, two studies presented data collected over different time periods (Extended data: Supplemental Table 284). A UK study reported the total costs incurred in the year before an HF event and in the 36 months after the event87, and a US study reported total Medicare payments over 30 days for patients discharged from hospital, grouped based on dyspnoea severity88.\n\nEight studies, all from the USA, compared costs and resource use for patients with HF with those for individuals without HF, or with other chronic diseases. Total annual costs were significantly higher for patients with HF than for those without HF (p<0.001): 4.6 times higher (US$27 152 vs US$5952) during 2010–2011, and 4.3 times higher during 2002–2011 (US$23 854 vs US$5511)79. A breakdown of constituent costs in this study is shown in Figure 5. Another study compared allowed monthly Medicare costs and resource use for patients with HF and all individuals who made Medicare claims (fee-for-service population). Per-patient allowed monthly costs for HF were 3.2 times higher than those incurred by the average fee-for-service patient (US$3395 vs US$1045)89. Patients with HF also had comparatively higher rates of inpatient admission, 30-day readmission, and use of skilled nursing facilities, meaning that higher costs were incurred for all of these resources, compared with the Medicare fee-for-service population.\n\nThis figure provides a graphical representation of annual healthcare expenditure with constituent costs for individuals with or without HF between 2002 and 2011. HF, heart failure.\n\nAdditional evidence indicated that HF is associated with increased resource use. A study of patients who were admitted to hospital for trauma and subsequently discharged found that the presence of HF was associated with a 71% increased risk of 30-day hospital readmission (24% vs 14%; p<0.0001)90. An examination of the resource use associated with hospital discharge for patients with HF, compared with all patients discharged, showed that costs for cardiology, supplies and devices, coronary care, and operating room services were 1.3–3.3 times higher for patients with HF91. Although few studies focused on indirect costs, one study examining care requirements was identified. In patients aged over 50 years, individuals with HF were significantly more likely than those without HF to make use of either formal (paid) care (HF: 9.1%; no HF: 1.5%; p<0.001) or informal care (HF: 33%; no HF: 8.6%; p<0.001), defined as care provided by a family member or unpaid volunteer92. Patients with HF required significantly more informal care per week than those without HF (32.1 hours vs 25.1 hours; p=0.002).\n\nThree studies compared the costs associated with HF and those for other chronic diseases. One study showed that similar total annual costs were associated with HF and obesity (US$1642 and US$1908, respectively); however, these conditions were considerably more costly than hypertension, which was associated with costs of US$431 per annum93. A second study reported that total annual costs were nearly twofold higher for HF than for DM, partly owing to higher inpatient and outpatient costs81. Finally, a study reporting disease-specific costs showed that HF incurred higher annual medical and pharmacy costs than asthma, coronary artery disease, COPD, DM, hyperlipidaemia, and hypertension76.\n\nThree studies showed that HF is associated with additional costs in patients with DM. A UK study estimated that annual inpatient care costs for a typical 60-year-old man with type 2 DM are more than six times higher in the presence of HF than if HF does not occur (£3191 for HF vs £459 for no HF)94. The occurrence of HF in a patient with DM was also estimated to result in 84% higher non-inpatient costs. The cost of preventable hospitalization for HF in patients with DM was reported as US$7949 in a US study95, and a second US study found that acute care for an HF episode incurred costs of US$23 758 per event-year, plus US$1904 in ongoing management costs96. The impact of HF in obese patients was also examined in one study. Patients with obesity but no HF incurred annual total costs of US$1908; however, this rose to US$5276 in patients with HF as well as obesity93.\n\nTwo US studies reported the cost impact of comorbidities in patients with HF (Extended data: Supplemental Table 284). Shaya et al. estimated the annual cost savings that could be made via a 20% reduction in a range of comorbidities, the most costly of which were cardiovascular disease and DM97. Smith et al. estimated the contribution of various comorbidities to the overall cost of care for patients with HF, finding that DM incurred the greatest costs of the comorbidities examined in the study98.\n\nOnly one study, conducted in Japan, reported costs by HF severity99. Hospitalization costs were modelled across NYHA classes: compared with NYHA class II, classes III and IV were associated with an additional US$490 and US$640, respectively (p<0.001).\n\nTwo Swedish studies collected data on the total annual direct costs associated with HFrEF100 and HFpEF101. These studies, however, were not conducted simultaneously or designed to compare costs by ejection fraction, so limited inference should be drawn from across-study comparisons. HFrEF was associated with slightly higher annual costs than HFpEF (€12 447 vs €11 344), as a result of higher costs associated with inpatient care, hospitalizations, and medication. Annual outpatient clinic costs were approximately 43% higher for HFpEF than for HFrEF (€1561 vs €1094).\n\nTwo US studies compared resource use between patients with HFrEF and those with HFpEF. One study found no significant difference between the two groups in terms of 30-day hospital readmission rate. However, patients with HFrEF had a significantly greater length of stay (LoS) in hospital than patients with HFpEF (HFrEF: 10.9 days; HFpEF: 8.5 days; p=0.027)90. In a second study, there were no significant differences between groups in terms of adjusted 30-day readmission rates, annual hospitalization rate, LoS, or pharmaceutical dispenses. Although the absolute differences between cohorts were small, HFpEF was associated with significantly more outpatient visits (HFpEF: 21.5; HFrEF: 20.1; p<0.002) and ED visits (HFpEF: 3.24; HFrEF: 2.94; p<0.002) annually than HFrEF, as well as a significantly higher rate of readmission within 1 year (HFpEF: 58%; HFrEF: 55%; p=0.010)102.\n\nInpatient care and hospitalization were identified as major cost drivers in HF and were reported as the single largest contributor to costs in multiple studies across different geographies. There was relatively wide variation across studies in the percentage of costs contributed by inpatient care, which can be accounted for by differences between populations and disparities in the types of costs included in each study.\n\nIn total, five European studies presented inpatient care or hospitalization as a percentage of total or direct costs: one each from Germany71 and Italy75 and three from Sweden72,100,101. Inpatient care or hospitalization contributed 69–87% of total costs in these studies (Extended data: Supplemental Table 384). Ten studies reported relevant data from North America, of which eight were conducted in the USA77,79–82,89,103,104, one took place in Canada85, and one conducted analyses using data from a clinical trial with study centres in the USA, Canada, and France105. The percentage of total costs contributed by inpatient or hospitalization costs across these studies varied between 47%79 and approximately 87%100,103 (Extended data: Supplemental Table 384). Three studies included only patients who died from HF; however, the contribution of inpatient care to total costs in these studies fell within the overall range for all studies77,82,105. An additional US study reported the proportion of total claims for HF accounted for by inpatient claims (60.5%) and outpatient claims (39.5%)93. One additional study, conducted in China, reported that inpatient care contributed 66% of total costs (Extended data: Supplemental Table 384)86.\n\nA German study estimated the future costs of inpatient care for HF, predicting that the overall budget impact in Germany would be €1.80 billion in 2025, up from €1.27 billion in 2009106. This predicted increase arose from a 14% increase in the cost allowance per patient with HF in hospital and a 23% increase in the number of patients with HF in hospital.\n\nSeveral other studies reported inpatient or hospitalization costs (Extended data: Supplemental Table 284). Seven studies, two from the UK94,107 and five from the USA13,108–111, collected data on overall inpatient/hospitalization costs associated with HF; however, these were not reported as a percentage of total costs. Additionally, five US studies reported the costs associated with a single hospitalization112–116, and one reported the cost of an ‘acute HF hospital episode’ as US$10 775117.\n\nPatients’ risk of hospitalization for HF or readmission within 1 year is likely to be influenced by factors such as disease severity, and consequently a wide range of rates were reported across the studies identified. There was a larger evidence base for all-cause 30-day readmission rates; these studies indicated that between 15% and 30% of patients hospitalized for HF are likely to be readmitted within a month after discharge from hospital.\n\nSeven studies73,74,76,81,103,105,118 reported 1-year hospitalization rates (Extended data: Supplemental Table 484). An Italian study found that HF-related and all-cause hospitalization rates were considerably lower in patients who had never been hospitalized for HF than in those who had (HF-related: 0.4% vs 22%; all-cause: 15% vs 59%)74. All-cause hospitalization was 30.8% in a Spanish study73. In the USA, HF-related hospitalization rates ranged from 6%76 to 22%103, and all-cause hospitalization rates ranged from 33% to 57%103. In a multinational study, 38% of patients experienced all-cause hospitalization105. A study in China reported relatively high rates of HF-related hospitalization within 1 year, at 34.8%118. A further 13 studies reported data on hospitalizations (Extended data: Supplemental Table 284)72,89,100,101,107,109,110,115,116,119–122, but did not present data as a percentage of patients experiencing hospitalization, did not report rates over 1 year, or included only patients who died of HF.\n\nStudies that reported 1-year hospital readmission rates, of which five were from the USA80,102,112,123,124, and one each from Italy75, Australia125, and China86, are summarized in Extended data: Supplemental Table 584. HF-related readmission rates ranged from 13.8% in a US study124 to 46.1% in the Italian study75, and all-cause readmission rates ranged from 55% in the USA102 to 73% in Australia125.\n\nHospital readmission rates at 30 days were reported in studies from the USA, Australia, and China (Extended data: Supplemental Table 684). In the USA, HF-related rates of readmission were 6.4–12.5%88,114,115,123,126,127, and all-cause rates were 17.1–30.4%80,88–90,102,108,113–115,123,126–132. An additional US study reported all-cause 30-day readmission rates after hospital discharge following implantation of an LVAD as 27.6%133. Similar rates were reported in Australia (HF-related: 11%; all-cause: 27%)125 and slightly lower rates reported in China (HF-related: 4.1%; all-cause: 16.2%)86. In addition, a study from Spain reported readmission rates in the 90 or 180 days after discharge from hospital (Extended data: Supplemental Table 284)134.\n\nTwo US studies focused on preventable readmissions. Gunadi et al. reported results from a care programme intended to reduce readmission rates, showing that the decrease in variable cost for each avoided hospital readmission was US$5652135. Chen et al. examined the rate of preventable hospital readmissions based on patients’ location and found that those in remote rural areas had a 27% lower risk of 30-day preventable readmission than patients living in urban areas136.\n\nA small number of the studies identified reported data that did not fall into any of our key areas of interest (Extended data: Supplemental Table 284). A Spanish study reported prescription drug costs137, a study from the Netherlands reported care home and nursing home costs for patients at the end of life138, and three North American studies reported hospital LoS52, nursing contacts139, and the association between heart rate and medical costs in patients with HF140, respectively.\n\n\nDiscussion\n\nThe evidence identified in this SR illustrates that HF is associated with a substantial humanistic burden. Patients with HF experience worse HRQoL than both individuals without HF and patients with other chronic diseases, and the burden of HF is greater for patients with more severe disease than for those at earlier stages. Poor mental health, comorbidities, and hospitalization are likely to be associated with worse HRQoL in patients with HF, whereas appropriate disease intervention can help to bring about improvements in HRQoL. This is supported by evidence that HRQoL decreases over time in the absence of disease management, but for patients who were provided support, HRQoL increased over time. Taken together with evidence indicating the positive impact of disease management and self-care, and structured support, this indicates possible ways of improving HRQoL for patients with HF.\n\nThe majority of the economic studies identified in this SR were conducted in the USA, with a smaller evidence base in Europe and relatively few studies from the rest of the world. HF is associated with considerable healthcare costs, and patients with HF incur higher costs and greater resource use, including inpatient, outpatient, and informal care, than individuals without HF or with other chronic diseases. The occurrence of HF as a comorbidity is also associated with extra costs. There is evidence to indicate that costs rise with increasing severity of HF, although this is based on only one study. The results of studies reporting costs and resource use by ejection fraction were mixed and did not provide sufficient evidence to conclude whether HFrEF or HFpEF incurs higher costs. Patients with HF are at a relatively high risk of hospitalization or readmission; accordingly, inpatient care and hospitalizations were identified as key cost drivers.\n\nSeveral studies suggested that improved disease management, with the aim of reducing the number of inpatient cases and reducing patients’ risk of hospital readmission, could be a way to limit the economic impact of HF. Specific strategies discussed included use of case-management programmes, identification of risk factors for readmission, and optimization of medication, for example by improving adherence.\n\nThis SR identified a broad range of studies; consequently, this review has focused on key areas of interest that best illustrate the impact of HF. Some studies have been presented in less detail, either because they did not discuss these key themes or because the data reported were not readily comparable with the rest of the evidence base, owing to differences in study population, setting, or time frame.\n\nAlthough a large number of studies were identified, some gaps in the evidence base were apparent. In studies comparing patients with HF and individuals with other chronic conditions, disease severity in the individual patient populations was not specified, nor was the impact of treatment interventions over the period of the study. This is particularly relevant when comparing different diseases, for which the effect of treatment upon HRQoL may vary considerably. Further studies on HRQoL in patients with HF, particularly those assessing the incremental effects of the disease over time or with increasing severity, would be valuable as part of an overall approach to identify means of improving patients’ well-being.\n\nA relatively small number of studies that assessed the economic impact of HF included detailed clinical information. The majority of studies in this SR, particularly those from the USA, which was the country with the largest evidence base, identified patients with HF in administrative claims databases using International Classification of Diseases diagnosis codes. This meant that factors such as comorbidities, HF severity by NYHA classification, and disease history were not known for all patient populations. These factors can have a large impact on disease outcomes and associated costs; therefore, some of the variation between the costs reported in different studies is likely to be attributable to factors that are not recorded. Economic studies in which patients’ medical records are available might therefore be valuable to obtain more granular data on cost drivers in HF. In addition, there is little published information on the indirect costs associated with HF, with very few studies reporting data on informal care for patients with HF; thus, the wider societal impact of the disease is not immediately apparent in the literature.\n\n\nLimitations\n\nThe design and scope of this SR meant that it is possible that not all relevant evidence was identified. Restrictions were applied to the evidence identified in the initial searches, by patient numbers, HRQoL instrument, and type of study. In particular, the restriction to economic studies including at least 100 patients and humanistic studies including at least 200 patients may have skewed the SR toward studies using patient registries and administrative claims data. Therefore, any studies with smaller sample sizes that included detailed clinical data for patients would not have been examined within the scope of the review. A formal assessment of the studies included was not carried out as part of the review process. This would typically include evaluation of each study’s inclusion criteria, measurement methods, analytic methods, and risk of bias141. Although all evidence discussed here is of value to address our research question, such an assessment might have highlighted any studies that were of particularly high or low quality, and helped to explain any major differences between the trends reported in different studies.\n\n\nConclusions\n\nOur findings indicate that patients with HF experience a greater humanistic burden and incur higher economic costs than individuals without HF or patients with other chronic diseases. There is some evidence to suggest that this burden increases further with worsening disease. Inpatient care and hospitalization costs were identified as key economic drivers.\n\nIt appears that slowing or preventing HF progression is likely to improve patients’ overall well-being, a healthcare aim that is particularly important given the substantial and increasing humanistic burden experienced by patients with HF. Reduction in patients’ hospitalization rates, and limiting the overall requirement for inpatient care, is the healthcare goal that would have the greatest impact on the economic burden of HF. The evidence that we have identified suggests that early treatment of HF to prevent or to delay disease progression, as well as careful disease management to avoid or lessen the need for hospital admission, is likely to lessen the humanistic burden and economic impact of HF.\n\n\nData availability\n\nAll data underlying the results are available as part of the article (included under extended data), and no additional source data are required to support our results.\n\nfigshare: Supplemental content 1 – Supplemental Table 1, http://doi.org/10.6084/m9.figshare.809991528.\n\nfigshare: Supplemental content 2 – Supplemental Tables 2–6, http://doi.org/10.6084/m9.figshare.809996984.\n\nThe systematic review protocol is available on request by contacting the corresponding author.\n\nPRISMA checklist: figshare: Supplemental Content 3 – PRISMA checklist, http://doi.org/10.6084/m9.figshare.8100020142.", "appendix": "Grant information\n\nThis study was funded by AstraZeneca. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMazimba S, Grant N, Parikh A, et al.: Heart failure performance measures: do they have an impact on 30-day readmission rates? Am J Med Qual. 2013; 28(4): 324–9. PubMed Abstract \| Publisher Full Text\n\nPatterson ME, Marken P, Zhong Y, et al.: Comprehensive electronic medical record implementation levels not associated with 30-day all-cause readmissions within Medicare beneficiaries with heart failure. Appl Clin Inform. 2014; 5(3): 670–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPerkins RM, Rahman A, Bucaloiu ID, et al.: Readmission after hospitalization for heart failure among patients with chronic kidney disease: a prediction model. Clin Nephrol. 2013; 80(6): 433–40. PubMed Abstract \| Publisher Full Text\n\nDunlay SM, Haas LR, Herrin J, et al.: Use of post-acute care services and readmissions after left ventricular assist device implantation in privately insured patients. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan der Plas AG, Oosterveld-Vlug MG, Pasman HR, et al.: Relating cause of death with place of care and healthcare costs in the last year of life for patients who died from cancer, chronic obstructive pulmonary disease, heart failure and dementia: a descriptive study using registry data. Palliat Med. 2017; 31(4): 338–45. PubMed Abstract \| Publisher Full Text\n\nRadhakrishnan K, Jacelon CS, Bigelow C, et al.: Association of comorbidities with home care service utilization of patients with heart failure while receiving telehealth. J Cardiovasc Nurs. 2013; 28(3): 216–27. PubMed Abstract \| Publisher Full Text\n\nDeVore AD, Schulte PJ, Mentz RJ, et al.: Relation of elevated heart rate in patients with heart failure with reduced ejection fraction to one-year outcomes and costs. Am J Cardiol. 2016; 117(6): 946–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSanderson S, Tatt ID, Higgins JP: Tools for assessing quality and susceptibility to bias in observational studies in epidemiology: a systematic review and annotated bibliography. Int J Epidemiol. 2007; 36(3): 666–76. PubMed Abstract \| Publisher Full Text\n\nGiles L, Freeman C, Field P, et al.: figshare: Supplemental content 3 – PRISMA checklist. http://www.doi.org/10.6084/m9.figshare.8100020" }	[ { "id": "50955", "date": "19 Jul 2019", "name": "Michael McGee", "expertise": [ "Reviewer Expertise Cardiology", "perioperative medicine", "indigneous health", "cardiac implantable electronic devices" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 article is a systematic review of the economic and personal burden of heart failure. The authors have preformed an admirably and extensive task given the scope of the project. The size and scope of the project limits it's utility, given the lack of bias assessment for each article and lack of clarity comparing outcomes and healthcare costs across vastly different healthcare systems.\nIntroduction: Doesn't provide background on previous estimated or quality of life reviews regarding HF. Does not compare HF to those of any other chronic disease process (such as renal failure, respiratory disease etc.). Nor does it explain the significant differences in HFpEF and HFrEF from an economic perspective.\nMethods: Not registered prospectively.\nResults: Well presented given the wide reaching scope of the review.\n\nDiscussion: Reasonable discussion. Should be noted that patients with HFpEF have limited treatment options and they're major mortality drivers are usually non-cardiac.\n\nConclusions: The authors conclude \"Our findings indicate that patients with HF experience a greater humanistic burden and incur higher economic costs than individuals without HF or patients with other chronic diseases\" and this conclusion is outside the reach of the review and would be better worded: \"Our findings indicate that patients with HF experience a greater humanistic burden and incur higher economic costs than individuals without HF\" as there is insufficient scope to comment on other chronic diseases.\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": "5794", "date": "19 Aug 2020", "name": "Bernt Kartman", "role": "Author Response", "response": "We would like to thank Dr McGee for taking the time to read and review our article and for his valuable contributions. We have taken on board Dr McGee’s feedback and implemented it in the following ways: We have distinguished between heart failure with reduced ejection fraction (HFrEF) and heart failure with preserved ejection fraction (HFpEF) in the introduction, noting the differences in aetiology and disease progression. We have also introduced discussion of the limited treatment options in patients with HFpEF compared with those with HFrEF We thank Dr McGee for noting that a risk of bias assessment was not performed and the review was not prospectively registered, and that this is a limitation of our research. We have highlighted this in the methodology section and would like to note that the protocol is available on request from the authors We have introduced the negative impact of HF on patients’ HRQoL relative to other chronic diseases and health individuals as well as the importance of surveying and quantifying the impact of heart failure on the patient using patient-reported outcomes. With regard to the conclusions drawn, we identified some evidence that compared HRQoL in patients with HF and those with chronic disease; however, we agree that this was not sufficient to support our conclusions and we amended them as you suggest We have also clarified that the wide range of outcomes and healthcare costs identified in our review limits the ability to draw to a clear conclusion from the evidence presented" } ] }, { "id": "62538", "date": "18 May 2020", "name": "Gerhard Wikström", "expertise": [ "Reviewer Expertise I am a collaborator in two referred papers in the reference list" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe results are well presented and the underlying material is impressive. This is a paper dealing with the quality of life and costs for patients with heart failure the data are extracted from databases, three types. Economic burden is readily understood but “humanistic burden”, how is that measured and defined? The study refers to health-related quality of life measurements of three different types. In total 124 studies were found dealing with the two types of outcomes that the study was looking for. Prognosis, quality of life, and costs relevant parameters for patients with heart failure. All three are well known and with discouraging results in several previous studies. The novelty in this paper is debatable since no new conclusions are drawn from the results. Only passive observations so common in this type of statistical work.\nWhy do the authors use the expression humanistic burden? It is not well defined and can be substituted for suffering, mental stress, low QoL etc. The last sentence in the first part of the discussion should be emphasized in order to justify the amount of statistics and work put down in this paper. In the discussion part about cost and resource use, the authors conclude with suggestions on how to reduce the increasing cost for HF management. Both the conclusions for increasing QoL and how to reduce the costs are very similar. Should that not stimulate the authors for more distinct conclusions about the interpretation of their 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? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [ { "c_id": "5795", "date": "19 Aug 2020", "name": "Bernt Kartman", "role": "Author Response", "response": "We would like to thank Professor Wikström for taking the time to read and review and for his valuable contributions. We have taken on board Professor Wikström’s feedback and implemented it in the following ways: We have further defined the term humanistic burden as used in the context of this article both in the introduction and in the discussion In the discussion, we have highlighted that, given the breadth of this review, a formal assimilation of the data identified is challenging and that we have therefore provided a narrative overview of the evidence identified, which was intended to supplement other published reviews We have amended the conclusions to include the challenges of drawing specific conclusions from such wide-reaching studies and provided some overarching conclusions; however, given the disparate nature of the evidence base, care should be taken in drawing definitive conclusions" } ] } ]	1	https://f1000research.com/articles/8-859
https://f1000research.com/articles/9-573/v1	08 Jun 20	{ "type": "Research Article", "title": "Effects of paracetamol (acetaminophen) on gene expression and permeability properties of the rat placenta and fetal brain", "authors": [ "Liam M. Koehn", "Yifan Huang", "Mark D. Habgood", "Kai Kysenius", "Peter J. Crouch", "Katarzyna M Dziegielewska", "Norman R. Saunders", "Liam M. Koehn", "Yifan Huang", "Mark D. Habgood", "Kai Kysenius", "Peter J. Crouch", "Katarzyna M Dziegielewska" ], "abstract": "Background: Paracetamol (acetaminophen) is widely used in pregnancy and generally regarded as “safe” by regulatory authorities. Methods: Clinically relevant doses of paracetamol were administered intraperitoneally to pregnant rats twice daily from embryonic day E15 to 19 (chronic) or as a single dose at E19 (acute). Control samples were from un-treated age-matched animals. At E19, rats were anaesthetised, administered a final paracetamol dose, uteruses were opened and fetuses exposed for sample collection. For RNA sequencing, placentas and fetal brains were removed and flash frozen. Fetal and maternal plasma and cerebrospinal fluid were assayed for ⍺-fetoprotein and interleukin 1β (IL1β). Brains were fixed and examined (immunohistochemistry) for plasma protein distribution. Placental permeability to a small molecule (14C-sucrose) was tested by injection into either mother or individual fetuses; fetal and maternal blood was sampled at regular intervals to 90 minutes. Results: RNA sequencing revealed a large number of genes up- or down-regulated in placentas from acutely or chronically treated animals compared to controls. Most notable was down-regulation of three acute phase plasma proteins (⍺-fetoprotein, transferrin, transthyretin) in acute and especially chronic experiments and marked up-regulation of immune-related genes, particularly cytokines, again especially in chronically treated dams. IL1β increased in plasma of most fetuses from treated dams but to variable levels and no IL1β was detectable in plasma of control fetuses or any of the dams. Increased placental permeability appeared to be only from fetus to mother for both 14C-sucrose and ⍺-fetoprotein, but not in the reverse direction. In the fetal brain, gene regulatory changes were less prominent than in the placenta of treated fetuses and did not involve inflammatory-related genes; there was no evidence of increased blood-brain barrier permeability. Conclusion: Results suggest that paracetamol may induce an immune-inflammatory-like response in placenta and more caution should be exercised in use of paracetamol in pregnancy.", "keywords": [ "placenta", "transfer", "inflammation", "permeability", "interleukin-1β", "IL1β", "⍺-fetoprotein", "AFP", "immune response" ], "content": "Abbreviations\n\nAFP, α-fetoprotein; CSF, cerebrospinal fluid; DPM, disintegrations per minute; E, embryonic (note that by longstanding convention all gestational ages in rodents are referred to as embryonic, but in this study E19 is a fetal stage); IL1β, Interleukin 1β cytokine; i.p., intraperitoneal; i.v., intravenous; P, postnatal; RNA-Seq, RNA sequencing; SD, standard deviation; µCi, micro Curie.\n\n\nIntroduction\n\nParacetamol (acetaminophen) is commonly taken, either by prescription or self-medication, for the relief of pain and fever. In pregnancy it is the most widely consumed drug, with estimates of expectant mothers talking this medication ranging from 56% in Australia and the Americas (Wyszynski & Shields, 2016) to 76% in Europe (Dreyer et al., 2015). The Australian Medicines Handbook (2019) states without qualification that paracetamol is safe for use in pregnancy and breast-feeding. However, epidemiological reports of behavioural effects in the offspring of mothers who took paracetamol during pregnancy are beginning to be published, suggesting a more cautious approach would be appropriate (see Bauer et al., 2018 and Discussion).\n\nIn a recent study we have found that paracetamol, when administered to a pregnant dam at doses within the clinical range used in patients, transfers across the placenta to reach the fetus at about 40% of the levels of the drug in the maternal circulation (Koehn et al., 2019b). Thus, the placenta provides a degree of protection for the developing fetus but the mechanisms involved are not yet understood, nor are the effects that paracetamol may have on placental functions. We have therefore carried out an RNA sequencing (RNA-Seq) study of E19 placentas and brains from control (un-treated) rats and from rats treated with a single (acute) or multiple (chronic) doses of paracetamol. This RNA-Seq study yielded the unexpected outcome of widespread up-regulation of inflammatory and immune-related genes in the placenta of the dam exposed to paracetamol over a prolonged period, with a much less pronounced effect on inflammatory-related genes following a single dose; however, many other genes showed a regulatory response following a single dose of paracetamol. Inflammatory responses during pregnancy have been linked to a range of clinical complications including pre-term birth, fetal cardiac conditions and neurological deficiencies (Challis et al., 2009; Huleihel et al., 2004; Romero et al., 2007; Salafia et al., 1989). High cytokine levels in blood have been linked to increased blood-brain barrier permeability (Anthony et al., 1997; Stolp et al., 2005a) and possibly leading to a range of health complications (Brochu et al., 2011; Nelson et al., 1998; Thornton et al., 2017). Inflammation in the placenta has also been linked to increased placental permeability, as shown in studies that identified a size-dependent increase in maternal-fetal nanoparticle transfer in mice (Tian et al., 2013).\n\nIn the present study, the inflammatory response in the placenta and the fetal brain following maternal paracetamol exposure was examined to see if it was associated with alterations in placental and blood-brain barrier permeability. Placental permeability was assessed using a low-molecular weight, hydrophobic molecule sucrose to determine the transfer in both directions: from the dam’s circulation to the fetal circulation and from the fetal circulation back to the dam. Transfer of a large molecule, the endogenous fetal-derived plasma protein α-fetoprotein (AFP), across the placenta into maternal circulation was also investigated. Results from both of these markers indicate that placental transfer was potentially affected by paracetamol treatment, and demonstrated increased levels of AFP detected in blood plasma of dams treated with paracetamol. The inflammatory cytokine IL-1bß was measured in fetal and maternal plasma; it showed higher levels only in fetal plasma following maternal paracetamol treatment. Permeability of the fetal blood-brain barrier to both small (sucrose) and large (plasma protein) molecules was not affected in spite of increased IL-1ß levels in fetal circulation. The results presented here highlight responses to paracetamol use during pregnancy that appear to be tissue-specific and dependent on duration of treatment. The results are discussed in the context of the appropriate use of paracetamol during pregnancy.\n\n\nMethods\n\nThe animals used in this study were the Sprague Dawley strain of Rattus norvegicus. All animal experimentation was approved by the University of Melbourne Animal Ethics Committee (Ethics Permission AEC: 1714344.1) and conducted in compliance with Australian National Health and Medical Research Guidelines. All animals were assessed as healthy prior to commencement of experiments. Animals were monitored prior to and following every injection ensuring there was no abnormalities in weight (>10%), appearance (fur) or behaviour (vocalisation, respiration, movements). All efforts were made to ameliorate any suffering of animals. They were handled by experienced researchers in such a way as to minimise stress prior to being anaesthetised.\n\nThese were supplied by the University of Melbourne Biological Research Facility and subjected to a 12 hour light/dark cycle with ad libitum access to food (dry pellets of a fixed formulation diet for laboratory rats and mice fortified with vitamins and minerals to meet the requirements of breeding animals after the diet is autoclaved or irradiated, supplied by Speciality Feeds, Western Australia) and water. Animals were housed in groups of 2–4 (adult) per cage (25cm x 35cm x 25cm on Breeders Choice paper bedding, made from 99% recycled paper; it is biodegradable with no added chemicals). Age groups investigated (at treatment completion) were embryonic day 19 (E19) pups of both sexes and dams, which were all primigravida 350–400g body weight) and non-pregnant female adults (175-230g body weight). E19 was chosen because this is a stage of development when adequate volumes of blood and cerebrospinal fluid (CSF) can be obtained for analysis from fetal rats without pooling (Dziegielewska et al., 1981) and individual pups can be injected intraperitoneally while still inside the uterine horn and kept viable for periods of time. Animal numbers were based on previous experience of such experiments and were the minimum number required to detect a significant difference between groups at p <0.05. Animals were selected for treatment groups to ensure weights were statistically similar between direct comparisons. Where possible, equal numbers of male and female fetuses were used. Animals on gestational day E19 were allocated to experiments by animal house staff, who had no knowledge of the particular experiments to be performed. The experimenters had no role in the selection of the animals, thus avoiding selection bias. The numbers (n) of animals used for each experiment are indicated in the relevant Methods or Results section and where appropriate in legends. Two litters in the sucrose permeability studies were excluded from the study. One mother died under anaesthesia. In the other case the fetuses were observed to be in poor physiological state, which would have affected the results.\n\nParacetamol (acetaminophen ≥99.0%, Sigma-Aldrich) was applied either at a high dose of 15mg/kg (higher limit in the range used clinically, Australian Medicines Handbook, 2019 and Koehn et al., 2019b) or a dose in the lower clinical range of 3.75mg/kg. Paracetamol was dissolved in sterile 0.9% sodium chloride solution for injection. For passive permeability experiments [U-14C]-labelled sucrose (Amersham International, CFB146) was injected in sterile 0.9% sodium chloride solution. Details are described in our previous study (Koehn et al., 2019b). Estimates of protein (AFP) permeability were obtained from western blot analysis of fetal and maternal plasma, as described below.\n\nAll experiments took place between 09.00 and 15.00h. Placentas and fetal brains from dams subjected to three treatment regimes were analysed in this study (n=4 for each tissue from each dam).\n\n(i) an E15 pregnant dam was given an intraperitoneal (i.p.) injection twice daily with 15mg/kg of paracetamol (dissolved in sterile 0.9% sodium chloride solution) over four days. On the 5th day (E19) the dam was given a final injection of the drug. This experiment is referred to as “chronic”;\n\n(ii) an E19 dam was given a single i.p injection of 15mg/ml paracetamol and is referred to as “acute”; and\n\n(iii) an E19 untreated dam (referred as control).\n\nIn experiments (i) and (ii), 30 minutes after the last injection of the drug the tissue samples (placentas, fetal brains) were collected (n=4 for each dam).\n\nFor RNA-Seq analysis, placental tissue was sampled as a cross section of the chorio-allantoic placental disc, following removal of the externally attached umbilical and maternal circulatory vessels. Brain samples of the cortex were dissected out as described before (Koehn et al., 2019a). Samples were collected under RNase free conditions and immediately frozen in liquid nitrogen and transferred to -80°C for storage. RNA extraction was completed using the RNeasy Plus Mini Kits and QIAshredder (Qiagen, catalogue number 74134) for placenta and using the RNeasy Plus Micro Kits (Qiagen, catalogue number 74004) for fetal cortex, following manufacturers specifications. RNA quantity and purity were determined using a NanoDrop ND-1000 UV-VIS spectrophotometer (Thermo Scientific).\n\nRNA samples were transported on dry ice to the Australian Genome Research Facility (AGRF) in Melbourne for Illumina, next-generation sequencing. Runs were 100bp single reads, providing raw FASTAq data. Data were processed using the Galaxy platform and their online software packages (Afgan et al., 2018). Default parameters were used unless directly specified. Data were groomed using FASTQ groomer (Galaxy version 1.1.1) and checked for read quality using FASTQc read quality reports (Galaxy version 0.72). Alignment was conducted using HISAT2 (Galaxy version 2.1.0) using the reference genome for rat (rn6; accession number GCA_000001895.4) and the reverse strand setting. For transcript quantification and differential expression analysis, three different methods were employed. In the first, pathway transcripts were assembled with cufflinks (Galaxy version 2.2.1.2) using the reference annotation for rat RefGene (genome) obtained from UCSC Main. Relevant data were passed through Cuffmerge (Galaxy version 2.2.1.1) and analysed for differential expression between groups of interests using Cuffdiff (Galaxy version 2.2.1.5). For the second and third pathway, counts were aligned using HTSeq-counts (Galaxy version 0.9.1) using the reverse strand setting. Generate Count Matrix (Galaxy Australia version 1.0) produced a matrix form of the data, which were then fed through either DEseq2 (Galaxy version 2.11.40.6) or EdgeR (likelihood ratio; Galaxy version 3.24.1) to receive differential expression analysis between treatment groups. Statistically different expression levels between relevant treatment groups were selected if present in two of the three datasets above the statistical threshold of p <0.05 for the adjusted P value of Cuffdiff (Padj), DEseq2 (q value) or EdgeR (FDR). This method of statistical selection minimizes the known false positives and false negatives that can be obtained due to the analysis pathway selected, ensuring all results can be found between multiple pipelines (see Seyednasrollah et al., 2016; Soneson & Delorenzi, 2013). Gene synonym names were produced via bioDBnet (Mudunuri et al., 2009). Pathway analysis was conducted using DAVID Bioinformatics Resources (version 6.8), with benjamini false discovery rate correction (Huang et al., 2009a; Huang et al., 2009b).\n\nIL1β cytokine concentrations in rat plasma were determined using ELISA specific for rat IL1β (R&D systems, Quantikine kit, catalogue number RDSRLB00, monoclonal mouse anti-rat IL1β) following the manufacturer’s protocol. Plasma samples were diluted 1:2 and 50μL of each sample was added to the same volume of assay diluent. Standard dilutions were assayed in duplicate. The plate was incubated at room temperature for two hours, then washed extensively. 100μl of rat IL1β conjugate was added and incubated for a further two hours. After additional thorough washes, the plate was incubated for 30minutes in 100μL of substrate solution then developed with 100μl of stop solution. Plates were read using a FlexStation 3 Multimode Microplate Reader (wavelength 450nm, using 570nm to correct for any optical imperfections in the plate) within 30 minutes of the addition of the stop solution. Cytokine concentrations were determined by comparison with the standard curve produced from each run.\n\nAll permeability experiments were conducted on E19 dams and fetuses. Two chronic paracetamol treatment regimes were used. Time-mated E15 pregnant dams were injected i.p. twice daily with either a 15mg/kg (referred to as “high”) or 3.75mg/kg (referred to as “low”) dose of paracetamol (dissolved in sterile 0.9% sodium chloride solution) over four days (“chronic” experiments). On the 5th day, at E19, these were compared to age-matched animals that were not treated (controls). Numbers (n) of individual experiments are indicated below and are included in the legends of corresponding figures in the Results section.\n\n14C-sucrose permeability. Animals were treated either with a “low” dose (3.75mg/kg) or “high” dose (15mg/kg) of paracetamol over four days starting at E15 following the same protocol as above. On the 5th day the pregnant dams (E19) were anaesthetised i.p. with 25% w/v urethane, (Sigma, 1ml per 100g body weight) and placed supine on a 35°C heating plate and an endotracheal cannula inserted prior to sampling. Left femoral artery and vein were cannulated. All injections were by slow infusion to the femoral vein; the cannula was flushed with 2ml of heparinized (Hospira Inc, 5000 units per ml) saline. Maternal blood samples were taken from the femoral artery; blood volume was maintained by intraarterial injection of equivalent volumes of 1ml heparinized sodium chloride solution. Blood (right cardiac ventricle), CSF (cisterna magna) and brains (cortex) were sampled from each fetus. Sampling was concluded when the state of the placental circulation (normal condition: umbilical veins pink with oxygenated blood) was deemed insufficient, usually around 90 minutes (see Koehn et al., 2019b for details). CSF samples were examined microscopically for traces of red blood cells and discarded if contaminated (Habgood et al., 1992). Maternal blood was also collected at the end of the experiment. Blood samples were centrifuged (5000rpm, five minutes). Plasma supernatant was removed and stored at -20°C until used.\n\nTwo sets of permeability experiments were conducted:\n\n(i) Fetal to maternal placental barrier permeability: pregnant animals treated with paracetamol as described above and control, untreated dams were terminally anaesthetized and an arterial cannula inserted into maternal circulation. Once the uterine horns were exposed, individual fetuses still within their amniotic sacs were injected serially with 14C-sucrose as described in Koehn et al. (2019b). Each fetus was taken at 30 minutes post injection. Maternal blood samples were collected at the same time as fetuses were consecutively removed for blood sampling. Maternal to fetal plasma levels ratios of 14C-sucrose were used as a measure of fetal to maternal placental transfer and calculated as follows:\n\n\n\n\n\nOne control litter (n=6); one litter from a chronically treated dam with a low dose 3.75mg/kg (n=5) and two litters from two chronically treated dams with a high dose (15mg/kg, n=5 for each litter) were used.\n\n(ii) Maternal to fetal placental barrier permeability: pregnant animals treated with paracetamol as described above and control untreated dams were terminally anaesthetized and 14C-sucrose was infused into the maternal circulation as detailed for paracetamol permeability studies above. Fetal samples were taken serially between 30 minutes and 90 minutes post injection. Blood samples from individual fetuses were collected together with time-matched maternal blood samples (Koehn et al., 2019b) and processed for liquid scintillation counting (see below) to obtain fetal/maternal plasma concentration ratios using the equation:\n\n\n\n\n\nOne control litter (n=8) and one litter from a chronically treated dam with high dose (15mg/kg, n=6) were used.\n\nPermeability of a fetal specific protein, AFP- western blotting. Levels of AFP in both the maternal blood samples and in fetal samples obtained from experiments of paracetamol treated dams as described above, were estimated using western blotting and antibodies to human AFP (DAKO).\n\nAll plasma samples were diluted 10-fold in isotonic saline (0.9%) prior to sample preparation. Samples were run using a total of 9µL of dam and 2µL of diluted fetal sera, denatured in 4x sample buffer (62.2 mM Tris, 5% (v/v) glycerol, 2% (w/v) SDS, and 0.0025% (w/v) bromophenol blue), heated to 95°C for five minutes and centrifuged briefly to remove potential particular matter. 12µL of each sample was loaded onto a 4–12% NuPAGE Novex Bis-Tris Midi gel (Life Technologies) and proteins were resolved at 200V for 40 minutes immersed in MES SDS running buffer (Life Technologies). Gel-resolved proteins were transferred onto PVDF membranes using iBlot gel transfer stacks (iBlot 2; Life Technologies) as per manufacturer's instructions. Membranes were incubated for one hour at room temperature in PBS-T blocking buffer (PBS supplemented with 0.05% (v/v) Tween-20 [Chemsupply]) and 5% (w/v) skim milk powder. Membrane was incubated with AFP primary antibody (AFP, rabbit polyclonal, 1:1000, DAKO, catalogue number A0008, RRID AB_2650473) diluted in the blocking buffer and incubated overnight at 4°C. After three PBS-T washes, the membrane was incubated for two hours at room temperature in horseradish peroxidase-conjugated goat anti-rabbit IgG (Cell Signaling; 1:5000, catalogue number 7074) secondary antibody. Immunoreactive protein bands were visualised by adding 1mL of Enhanced Chemiluminescence mixture (ECL Advance, GE Healthcare) onto membranes and detecting luminescence using a FujiFilm LAS-3000 imager at three and 75 second exposures. Densitometric quantitation of immunoreactivity was performed using ImageJ 2-bit, v1.46 run on OSx 10.14 Mojave on 8-bit TIFF file images. All samples that were directly compared were run on the same gel. Serum from an age-matched non-pregnant female was used as a negative control, while an E19 pregnant dam that was not injected with paracetamol was used as a positive control. Both samples were included on every gel.\n\nBlood-brain barrier permeability in the fetus was estimated using (i) radioactive sucrose as an example of a small molecular marker permeability and (ii) plasma protein (immunohistochemistry), as an example of a large molecular marker permeability (Habgood et al., 1993; Johansson et al., 2008; Stolp et al., 2005b). Fetal blood, CSF and brain samples were obtained from the same placental permeability experiments described above.\n\n14C-sucrose permeability. For estimation of transfer from mother to fetus, pregnant animals treated with paracetamol (as above) were anaesthetized i.p. with urethane. Starting at 30 minutes after the last maternal injection, embryos were individually extracted. For estimation of transfer from fetal blood to fetal brain and CSF, the fetuses were exposed and injected i.p. with 14C-sucrose.\n\nIn both types of experiment fetal blood and CSF were sampled as described previously (Koehn et al., 2019b). Fetal brain samples were taken by opening the cerebral hemispheres to expose the lateral ventricles and a sample of the parietal cortex was removed, taking care to avoid the choroid plexuses. Brain or CSF to plasma ratios of 14C-sucrose radioactive counts were used as an estimate of the transfer of sucrose across the blood brain barriers. These were calculated using the equation:\n\n\n\nTreatment groups investigated were control, no paracetamol (n=13), chronic low dose (3.75mg/kg, n=11) and chronic high dose (15mg/kg, n=11) in fetuses that were injected directly. In experiments in which the 14C-sucrose was injected into the treated mothers, numbers of pups used were control (n=8), acute (n=10) and chronic high dose (n=6).\n\nImmunohistochemistry. Individual fetal brains were fixed in Bouin’s fixative for 24–48h then dehydrated through graded alcohols, cleared in chloroform and embedded into paraffin wax blocks. These blocks were cut serially into coronal 5µm sections (Leica microtome). Selected sections were heated for 30 minutes (60°C) then washed twice with Histolene (Fronine) for 10 minutes, then five minutes. The sections were rehydrated through graded alcohols for five minutes each (100%, 100%, 95%, 70%) and washed in phosphate buffered saline with 0.2% Tween20 for five minutes. Peroxidase and protein blockers (DAKO) were added to sections and incubated at room temperature for two hours each to block non-specific binding. The primary antibody, plasma protein (anti-rat whole serum, SIGMA, catalogue number R5129, rabbit polyclonal) diluted 1:3000 in a blocker (0.5% fish gelatine and PBS + Tween20), was applied to the slides and incubated overnight at 4°C. After three washes of PBS + Tween20 for five minutes each, the secondary (swine anti-rabbit, DAKO, catalogue number Z0196, polyclonal) and tertiary antibodies (rabbit PAP, SIGMA, catalogue number P1291) both diluted 1:200 were each added and incubated for two hours at room temperature with washes between incubations. Sections were developed with DAB (Diaminobenzidine) using DAKO DAB+ kit (catalogue number K3468) according to manufacturer’s directions and washed in running water for five minutes. Sections were dehydrated through a series of graded alcohols (70%, 95% for five minutes, then 100% for 10 minutes), then 3x five minutes in histolene washes. Slides were then mounted using DPX mounting medium (Fronine). Stained sections were examined under a compound microscope (Olympus, BX50) fitted with a digital camera (Olympus DP70). One control slide was included with every round of immunostaining and had the primary antibody omitted but was otherwise treated in the same way. These were always blank. A total of 11 brains with at least two brains per treatment group were prepared and serially sectioned and mounted on glass slides. Each slide contained 6–8 sections, every 10th slide was stained with haematoxylin and eosin for general morphology. One or two adjacent slides per brain were immunostained for plasma protein from comparable brain regions.\n\nPlasma (10μL), CSF and every injectate (1μL of 1:10 dilution) were weighed and transferred into scintillation vials. In all experiments the radioactivity in the injectate was measured to confirm the uniformity of the injected material. Soluene350 (0.5ml, PerkinElmer) was added to the brain samples and incubated overnight at 36°C. Prior to measurement, two drops of glacial acetic acid (Sigma) were added to brain vials to neutralize the strongly alkaline Soluene350. All samples were mixed with 5ml of scintillation fluid (Emulsifier-safe, PerkinElmer) and measured on the liquid scintillation counter (Tri-Carb 4910 TR, PerkinElmer). Counting was conducted in disintegrations per minute (DPM) for five minutes each with luminescence correction on. Vials containing control, non-radioactive tissues processed identically were also counted simultaneously to establish background counts (which were subtracted from all radioactive samples). Counts were normalized to the sample weight and expressed as DPM per µL or µg of sample. Results are described as concentration ratios, defined as a % of the counts (per µL or µg) in the compartment of interest (brain, CSF, maternal or fetal plasma) divided by the counts (per µL) in the plasma compartment of comparison (see also Koehn et al., 2019b).\n\nRNA-Seq data analysis is detailed above, with significance set at p <0.05. For all other experimentation, statistical differences between treatment groups were determined by unpaired Student t-tests using Prism 6.2 (Graphpad Software Inc) with significance set at p <0.05.\n\n\nResults\n\nE19 placentas and brains from three treatment groups were compared for transcriptomic analysis using RNAseq datasets: (i) untreated controls (n=4), (ii) acutely paracetamol treated (n=4) and (iii) chronically paracetamol treated (n=4) dams (see Methods), providing a three-way comparison for each tissue (Figure 1 and Table 1–Table 5).\n\nTranscript numbers for Chronic/control, Acute/control and Chronic/acute comparisons. Controls were from untreated animals. For details of chronic and acute dosage schedules see Methods. Data derived from RNA-Seq analysis. Overlapping segments represent shared genes.\n\nTranscript numbers in placentas treated with paracetamol (chronic, acute or control, n=4 in each group). For details of dosage schedules see Methods. Data from RNA-Seq analysis. FC = fold change compared to control.\n\nUp-regulated and down-regulated inflammatory and immune-related gene changes in E19 placenta following no treatment (co, controls), acute (ac,) or chronic (ch) maternal paracetamol treatment; see Methods for details of dosage schedule. Data from RNA-Seq analysis. Numbers are fold changes for comparisons indicated (Ch/Co, Ac/Co, Ch/Ac). The chronic/acute comparison indicates significant differences in regulation between the two dosage regimes. In all cases expression was greater with chronic treatment.\n\nTranscript numbers following chronic, acute or control maternal treatment with paracetamol, n=4 in each group. For details of dosage schedules see Methods. Data from RNA-Seq analysis. FC = fold change compared to control.\n\nUp-regulated and down-regulated inflammatory and immune-related gene changes in the E19 fetal brain following no treatment (Co, controls), acute (Ac,) or chronic (Ch) maternal paracetamol treatment; see Methods for details of dosage schedule. Data from RNA-Seq analysis. Numbers are fold changes for comparisons indicated (Ch/Co, Ac/Co; Ch/Ac). Compared to the placenta, in the fetal brain many fewer inflammatory and immune-related genes showed regulatory changes and there were no significant differences between acute and chronic treatments. - indicates no significant difference in fold changes, not that there was no fold change.\n\nOnly genes that showed a response in placentas from E19 animals (left panels) and fetal brains (right panels) following both acute and chronic maternal treatment with paracetamol are shown; see Methods for details of dosage schedule. Data from RNA-Seq analysis. Numbers are fold changes for comparisons indicated (Ch/Co, Ac/Co; Ch/Ac). There were no significant differences for these genes between acute and chronic treatments, although there were small fold changes (data not shown).\n\nAs illustrated in Figure 1, following maternal exposure to paracetamol (either acute or chronic), there was a large number of genes that significantly altered their expression in the E19 placentas in two-way comparisons to control tissue, with much fewer that changed between the two treatment groups (chronic/acute). Most genes were uniquely regulated, either up or down, depending on treatment duration, with relatively few that were common to both treatment regimes (65 up-regulated and 57 down-regulated). In contrast, in a three-way comparison, only one gene, Nfkbia (NF-kappa-B inhibitor alpha), was shared in all three comparisons (Figure 1). NFKB is a transcription regulator that is activated by various intra- and extra-cellular stimuli such as cytokines.\n\nThe expression of 121 transcripts (the sum of up-regulated and down-regulated genes in the chronic/acute comparison) was significantly different between acute and chronic treatment groups, suggesting an effect of treatment duration. Of these genes, 34 were significantly up-regulated in chronically treated animals when compared to either the acute treatment group or the control group and eight were down-regulated (Figure 1).\n\nComparing datasets of placentas from chronically treated dams with untreated control dams, the expression of 737 genes was significantly different (either up or down) (Figure 2). The top 50 up-regulated and down-regulated genes in E19 placentas are displayed for both acute and chronic treatment groups compared to controls in Table 1. In the E19 placentas, many of the top genes up-regulated following chronic treatment were related to immune-response and inflammation (Table 1, Table 2 and Table 5). It is difficult to determine the extent to which a statistically difference in gene expression is also functionally significant. It is perhaps worth noting that fewer genes were up-regulated two-fold or more with either acute or chronic paracetamol treatment (25 and 34 genes, respectively) compared to the number that were down-regulated two-fold or more (58 and 61, respectively). In addition, the degree of down-regulation was appreciably greater for many of these genes compared with those that were up-regulated. This was particularly evident for the chronically treated group compared to the control group, with five genes down-regulated greater than 500-fold (Afp, apoc2, rbp4, apob and fgb, Table 1)\n\nTranscript numbers in placenta and brain from chronically (15mg/kg) treated compared to control, untreated animals. For details of chronic dosage schedule see Methods. Data derived from RNA-Seq analysis. Overlapping segments represent shared genes.\n\nGenes that showed a regulatory response in placentas of animals following both acute and chronic treatment with paracetamol are listed in Table 5. Seven of these down-regulated genes showed a fold change of more than two, which was greater in the chronically treated placentas. Other changes were so small that they are unlikely to be of much functional significance.\n\nIn the placenta of chronically treated rats there was a notable up-regulation of immune response related genes compared to the acutely treated group (Table 2). Figure 3 illustrates an analysis from biological Gene Ontology (GO) categories of immune response genes (A) subdivided into the innate (B) and adaptive (C) immune systems in the chronically treated animals. In the placenta these included GO biological processes such as neutrophil chemotaxis (p= 4.7E-05) and innate immune response (p=0.045). Figure 3 illustrates that the number of significantly up-regulated genes was substantially more than the number of down-regulated genes and that most of these were in the innate immune system category, with a small number in the adaptive immune system. A list of inflammatory and immune-related genes that were up-regulated in the placenta following chronic treatment is shown in Table 2. Overall, some 36 genes showed a statistically significant up-regulation. These included 15 genes that were upregulated two-fold or more. As can be seen from Table 2, the third most up-regulated gene in the placenta following chronic paracetamol exposure was Il1ß. Figure 4 illustrates the number of Il1ß gene transcripts in the three treatment groups in the fetal brain and placenta. There was a prominent increase in Il1ß transcripts in the placentas from the dams treated with chronic paracetamol and no difference between the datasets of placentas from the control and acutely treated mothers, both showing very low numbers. IL1ß is a prototypical marker for inflammation and immune response, with up-regulation in the chronically treated placenta of 13.3 fold change; it could thus be a potential indicator of fetal harm. The response in the placenta following a single acute dose of paracetamol was much more muted (Table 2).\n\n(A) “immune response”, (B) “innate immune system” and (C) “adaptive immune system”. The number of genes significantly up-regulated (green) and significantly down-regulated (red) are shown for adult brain, E19 brain and E19 placenta, as determined by RNA-Seq. Results are displayed for chronic and acute paracetamol treated rats (n=4). For details of chronic and acute dosage schedules see Methods.\n\nTranscripts per million in E19 fetal placenta and brain from control (n=4), acute (n=4) or chronically treated dams (n=4) as determined by RNA-Seq. (HTSeq-counts, EdgeR). Means ± SD. * p <0.05.\n\nAmongst the down-regulated genes in the placenta (Table 1) were several transcripts for plasma proteins (AFP, transthyretin and transferrin, see Discussion) that have been shown to down-regulate under inflammatory conditions (negative acute phase response, Heinrich et al., 1990; Hu et al., 2019; Mackiewicz et al., 1990). Two of these were markedly down-regulated in the acute experiments and further down-regulated in the chronic experiments (Table 1). This suggest that the response of these plasma protein genes was rapid in onset and continuing over several days in the presence of chronic treatment. In contrast, the up-regulatory response of cytokine genes only became prominent in the placentas of animals chronically exposed to paracetamol (Table 1; Figure 4).\n\nIL1ß concentration (ELISA). In order to see if the increase in transcript numbers for IL1ß in placentas from dams treated chronically with paracetamol (Figure 4) translated into an increase in its protein concentration, the levels of this cytokine in plasma of both the dams and pups were measured using a commercially available ELISA kit (see Methods). Results are illustrated in Figure 5. None of the dams in any of the treatment groups had a detectable level of IL1ß in their plasma (limit <5pg/ml). In contrast, detectable levels of IL1ß in the plasma of many of the E19 fetuses whose mothers had been treated with paracetamol were demonstrated, and the levels were generally higher in fetuses of mothers treated chronically.\n\nHowever, the levels of IL1ß were quite variable even within each treatment group. In the two groups of pups from dams treated with either dose of paracetamol (15mg/kg or 3.75mg/kg), several individuals had concentrations of the cytokine markedly increased, up to 100pg/ml (Figure 5). Overall, in dams treated with paracetamol, 19/39 fetuses had IL1ß levels above 5pg/ml.\n\nSamples from control (n=2/4), acute (n=2/4) or chronic paracetamol treated dams at low (3.75mg/kg, n=5/16) or high (15mg/kg, n=7/19) doses; n=dams/fetuses sampled. Measured by ELISA (R&D Quantikine). Means ± SD.\n\nTranscriptomic analysis of the E19 fetal brain was carried out in material collected from the same animals as was prepared for placental analysis, thus allowing a direct comparison between the response of the two tissues to paracetamol treatment of the mother.\n\nAs illustrated in Figure 1, following maternal exposure to paracetamol, there was a large number of genes that significantly altered their expression in the fetal brain.\n\nAs shown in Figure 2, comparing the dataset for fetal brains from chronically treated dams with untreated control dams, there was a total 1128 genes with significantly different transcript numbers in the E19 brain. The top 50 up-regulated and down-regulated genes in the E19 brain are shown for both acute and chronic treatment groups compared to controls in Table 3. Many more genes were up-regulated following acute treatment rather than chronic treatment. In contrast, more genes were down-regulated in the brains of chronically treated fetuses compared with the acutely treated. Additionally, the level of down-regulation was greater for most transcripts than up-regulation following both acute and chronic paracetamol treatment, for example Col1a1 (collagen type 1 alpha 1 chain) and Col3a1 (collagen type 3 alpha 1 chain), see Table 3. There will be a further analysis of the brain data in a later publication (Koehn et al., unpublished reports) that will deal with expression of ABC efflux transporters and related enzymes as these may play a role in the extent to which paracetamol enters the brain at different stages of development (Koehn et al., 2019b).\n\nIn addition to effects of the length of exposure to the drug on gene expression in individual tissues, the regulation in brain and placenta was very different following the same treatment.\n\nOnly 98 genes were significantly regulated in both tissues, equating to 5.5% of the transcripts that changed their expression (Figure 2).\n\nIn the E19 placenta many of the top genes up-regulated following chronic treatment were related to immune-response and inflammation, including Il1ß, which was 3rd highest (Table 1). In contrast, in the brain, very few transcripts for Il1ß (Figure 4) or other cytokines (Table 4) could be detected and there was no difference in transcripts for Il1ß between the treatment groups (Figure 4). Table 4 lists the inflammatory and immune-related genes that were up- or down-regulated significantly in the E19 brain. The changes were very small compared to the placenta in both the innate immune and the adaptive immune category (Figure 3). Table 5 shows immune/inflammatory related genes that showed a regulatory change in both the placenta and the brains from both acutely and chronically treated fetuses.\n\nNo changes in plasma protein transcript numbers were detected in the fetal brain (see Discussion). This, together with lack of up-regulation of the inflammatory cytokine Il1ß, as seen in the placentas, indicates that an inflammatory response was elicited by paracetamol in the placenta but little or none in the fetal brain. We do not have information if other organs not investigated in this study, such as the liver, could also have been affected.\n\nIn order to investigate if a prolonged exposure to paracetamol and resulting inflammatory response could affect the permeability of the placenta, two sets of permeability experiments were conducted using a small molecular size marker, 14C-sucrose (see Methods). These were designed to examine the transfer from the mother to the fetus but also from the fetus back to the mother. Results are illustrated in Figure 6 and Figure 7.\n\nFollowing maternal paracetamol treatment fetuses, still within their amniotic sacs, were directly injected (i.p.) with 14C-sucrose and plasma from both fetus and dam were collected to calculate maternal/average fetal plasma ratio (%) over time. Treatment groups: control (n=6), chronic low dose (3.75mg/kg, n=5) and chronic high dose (15mg/kg, n=5); n refers to number of fetuses.\n\nMothers were treated with paracetamol. 14C-sucrose was injected (i.p) into the mothers. Blood samples from individual fetuses were collected at the same time as maternal samples. Treatment groups were: untreated (control, n=8) and paracetamol injected (chronic dose 15mg/kg, n=6) dams. n refers to number of fetuses. Transfer calculated as fetal/maternal plasma ratio (%).\n\nFetal to maternal transfer of 14C-sucrose. To investigate the placental transfer of sucrose from fetus back to the dam following maternal paracetamol exposure, sucrose was injected directly into the pups still within their amniotic sacs (see Methods). Two litters were injected in mothers that had been treated with chronic high doses of paracetamol and one litter from a mother treated with chronic low dose paracetamol. These were compared with one litter from an untreated control mother. Plasma samples from both the fetuses and dam were collected and ratio of 14C- sucrose estimated (see Methods). The results are shown in Figure 6. All three of the litters from mothers treated with chronic paracetamol (either high or low dose) showed slightly higher permeability from the fetus back to the mother than in the control dam. However, the ratios are extremely low, making accurate comparison difficult.\n\nMaternal to fetal transfer of 14C-sucrose. In order to investigate if the rate of transfer of a small molecular marker from dam to fetus across the placental barrier was affected following paracetamol exposure, dams either untreated (control) or treated with chronic high (15mg/kg) doses of paracetamol were given a final intravenous (i.v.) injection of 14C-sucrose 30 minutes before removing their fetuses (Figure 7). Blood samples from dams were time matched to the removal and blood collection from each fetus (see Koehn et al., 2019b). The transfer from the mother to the fetus in the paracetamol treated dams was slightly less than that in the control animal. The much higher ratios obtained in the maternal to fetal transfer experiments (Figure 7) compared to the fetal to maternal transfer (Figure 6) are due to the differences in volume of distribution, hence dilution of the marker, when sucrose is injected into the mother or into the fetuses.\n\nDetection of AFP in fetal and maternal plasma. In order to investigate if exposure to paracetamol can also influence the transfer of a protein from the fetal circulation into the maternal blood across the placenta, western blot analysis was made of fetal and maternal plasma samples using cross-reacting antibodies specific for AFP (see Methods). Figure 8A shows the blot that contained both the fetal and maternal samples together with a negative control (non-pregnant female rat). Densitometry measurements are illustrated in Figure 8B together with maternal/fetal ratios. There was no detectable band in the control sample and all maternal samples showed a much lower level of the protein than fetal samples. The levels of the protein in fetal samples did not appear to change significantly between the control and any of the treatment groups (Figure 8B), but in maternal samples, AFP levels were higher in all treated dams compared to an un-treated control. This was reflected in the ratios of AFP in maternal to fetal plasma (Figure 8B, right panel), showing that even a single injection (acute) of paracetamol may increase placental transfer, while prolonged exposure to the drug increased AFP transfer by about three times compared to the control animals.\n\nA) Western blots of AFP in plasma from dams and fetuses in different treatment groups. Numbers for dam blots are samples from individual animals; numbers in fetal plasma blots indicate individual fetuses from corresponding dams. Treatment groups were: control (1/1b; n=1), acute (2/2b; n=2), chronic low dose (3/3b; n=2), chronic high dose (4/4b; n=3) and non-pregnant control (5; n=1). B) Estimations of AFP in dam and fetal plasma (densitometry units from blots in (A) and fetal to maternal transfer of AFP expressed as dam/plasma AFP ratio (%). Note: each point represents an individual animal. Note that all treated dams had higher plasma levels of AFP than the un-treated pregnant dam; AU are ordinate arbitrary densitometry units.\n\nTwo different molecular size markers (14C-sucrose and plasma proteins) were used to assess any changes in blood-brain barrier permeability following chronic paracetamol treatment of the dam. The samples were obtained from the same experiments as the placental permeability studies.\n\nTransfer of 14C-sucrose into the brain and CSF following different paracetamol treatment regimes. To investigate the transfer of 14C-sucrose into the fetal brain after maternal paracetamol exposure, fetal blood, brain and CSF samples from dams untreated (control) or treated acutely or chronically with either low (3.75mg/kg) or high (15mg/kg) doses of paracetamol were measured. As shown in Figure 9, there was no significant difference in the transfer into the brain and CSF between any treatment groups (Figure 9).\n\nFetuses were exposed to 14C-sucrose either directly (fetal i.p. injection) or indirectly (maternal i.v. injection). Treatment groups investigated were control, no paracetamol (n=13), chronic low dose (3.75mg/kg, n=11) and chronic high dose (15mg/kg, n=11) in fetuses that were injected directly. In experiments in which the 14C-sucrose was injected into the mothers; n numbers were control (n=8), acute (n=10) and chronic high dose (n=6) in the mothers. Means ± SD.\n\nEntry into the brain and CSF when the 14C-sucrose was injected directly into the fetus was also investigated and results are illustrated in Figure 9. Here too there were no significant differences in the entry of sucrose into brain and CSF between the three treatments.\n\nHowever, the fetuses that were directly exposed to sucrose (i.p injection) showed a lower level of transfer into the brain and CSF compared to those that were exposed indirectly (i.v. injection to dam), around 10% compared to 40%, respectively. This reflects differences in distribution volume following the different routes of injection as well as the time involved in samples collection.\n\nBlood brain barrier integrity for endogenous plasma protein. Transfer of large molecule plasma proteins into the fetal brain following maternal paracetamol exposure was studied using immunohistochemistry and antibodies to rat serum proteins (see Methods). Brains were matched with plasma samples containing detectable IL1ß levels as estimated by ELISA (Figure 5). The distribution of the proteins in E19 brains from control, acute and chronic high dose (15mg/kg) paracetamol treated dams is illustrated in micrographs in Figure 10. There was no evidence of a “leak” of protein in any of the vessels in the fetal brains examined. In all sections stained from all brains investigated, immunostaining was exclusively localised in the blood vessels, choroid plexus stroma and precipitated CSF and there was no visible difference in the brain morphology between treatment groups.\n\nA) Hematoxylin and eosin coronal section of E19 neocortex of fetus from mother treated with chronic high dose paracetamol. B) Adjacent section from same brain as A immunostained for plasma proteins. C) High power image from B (box). D. High power immunostained image of E19 neocortex of fetus from mother treated with acute high dose paracetamol. Note that all cerebral vessels appear intact with protein immunostained deposits all within blood vessel lumen, indicating that paracetamol treatment has not affected their barrier permeability to plasma proteins. Bars in A & B are 1mm; bars in C & D are 100µm.\n\nThus, the results clearly show that the blood-brain barrier, at least to plasma proteins and to sucrose, was not affected by paracetamol exposure of the dam, the inflammatory response in the placenta nor the increased levels on IL1ß in fetal blood.\n\n\nDiscussion\n\nIn order for a drug taken by a pregnant mother to reach the fetal brain it has to cross both the placental and the blood-brain barriers. Any changes to normal functioning of these interfaces could have detrimental effects on fetal health and pregnancy outcomes. We have therefore analysed the transcriptomic changes in rat E19 placentas and brains following paracetamol treatment of the dams. Paracetamol is one of the most commonly used medications in pregnancy (Dreyer et al., 2015; Wyszynski & Shields, 2016). Pregnant rats were treated with paracetamol acutely and chronically and compared to controls (no treatment). The doses used were within the clinically recommended range (0.5g to 4g in 24 hours in adults). In the case of the chronic treatment, this corresponded to a relatively prolonged period of pregnancy in the rat (E15-19, about 25% of gestation). This was followed by investigating placental transfer of small and large molecules from the dam to the fetus and from the fetus back to the maternal circulation, to see whether paracetamol exposure altered barrier function. Finally, the permeability of the blood-brain barrier was analysed in the fetuses of paracetamol treated and untreated dams.\n\nFrom the results it was apparent that some form of acute phase response was elicited as transcripts for several plasma proteins were down-regulated in placentas of both acute and chronic treated animals (Table 1). These proteins were AFP, transthyretin and transferrin (Vranckx et al., 1989), fibrinogen beta chain (Birch & Schreiber, 1986) and apolipoproteins Apoa1-4, several of which are known to respond to inflammation as negative acute phase proteins (Tu et al., 1987). Since a marked response was already apparent after a single dose of paracetamol, it seems that this was a rapid response to paracetamol, which was sustained and increased when the treatment was chronic. A summary of transcript numbers for AFP, transferrin and transthyretin, together with numbers for Il1ß for comparison, is presented in Table 6 for both the brain and the placenta. These clearly show that some form of acute phase response was taking place in the placenta following paracetamol treatment; however, other typical acute phase response-related cytokines were not up-regulated (such as TNFα or IL6). Transcript numbers in the brain did not change, demonstrating that the acute phase response was tissue specific and restricted to the placenta.\n\nIn placental samples transcript numbers for the three negative acute phase proteins were smaller in chronically treated animals compared to controls and all IL1β numbers were greater than in controls. There was some variation in values between individual placentas which was more obvious in samples from acutely treated animal indicating that the response was potentially time-dependent. In all brain samples the transcript numbers were very low, with no evidence of an inflammatory response. This indicates that the response in the placenta was tissue specific.\n\nSeveral immune and inflammatory-related genes were up-regulated in placentas of animals treated with the chronic dose regime, but much less so in the placentas of acutely treated animals (Table 1, Table 2 and Table 5). The key inflammatory cytokine, IL1ß, was shown to be present in the blood of a high proportion of fetuses of mothers exposed to both acute and chronic treatment with paracetamol. The levels were variable in different fetuses but generally higher in the chronically treated animals. No IL1ß could be detected in either the maternal blood of paracetamol treated animals or in fetuses of control untreated animals. This confirms that paracetamol was indeed eliciting an inflammatory response but only on the fetal side of the placental circulation. Thiele et al. (2015) reported that pregnant mice treated with either 50 or 250mg/kg paracetamol showed some immune responses in the uterus and some morphological changes in the placenta. However, they did not investigate possible immune responses in the placenta and the doses of paracetamol were much larger than the ones we used and were well above the clinical range.\n\nIn order to determine if prolonged paracetamol exposure of the dam could affect some aspect of placental function, we have estimated placental permeability to a small molecular marker, sucrose and to large plasma protein AFP in both directions i.e. from the dam to the fetuses and from the fetuses back to the dam. The results showed that there was a small and variable increase in permeability to 14C-sucrose and of AFP permeability in the direction from fetus to mother (Figure 6 and Figure 8). There may also have been a small decrease in sucrose permeability from mother to fetus (Figure 7) but due to small numbers this is inconclusive.\n\nPlacental inflammation induced by lipopolysaccharide (LPS) injection in pregnant rats has been reported to induce maternal serum and placental cytokines and increased maternal serum AFP (Hu et al., 2019). In those experiments LPS did not increase the expression of AFP in fetal liver, maternal liver or placenta, but did reduce the fetal serum AFP levels, a pattern implying a possibility of increased transfer of AFP from the fetus to the mother, thus depleting it from fetal circulation. We did not find any difference in fetal AFP levels but this discrepancy could be due to either the duration and severity of the response or sensitivity of the methods used.\n\nPermeability of the fetal blood-brain barrier to both sucrose and plasma protein was also investigated. In contrast to the placenta, there was no evidence of a change in brain barrier permeability to either marker in fetuses of dams treated with paracetamol. This is relevant to earlier studies in which inflammation induced by LPS was shown to result in a breakdown of the blood-brain barrier that was age-dependant (Stolp et al., 2005a; Stolp et al., 2005b). However, it is likely that E19 is at a developmental stage when the response to LPS is not yet developed, as shown in a similar study in a marsupial species, Monodelphis domestica (Stolp et al., 2005a).\n\nThe study has been carried out in pregnant rats at a single gestational age (E19). This stage of brain development in rats at E19 is approximately equivalent to 22–24 weeks gestation in humans (Clancy et al., 2001), corresponding to the earliest age of viability (Fischer et al., 2009; Stoll et al., 2010). The rat and human placentas are both classed as hemochorial (Blood et al., 2007; Dawe et al., 2007) but there are differences in morphology, in particular that the rat placenta has more morphological layers between the fetal and maternal circulations. However, that might mean that the relatively small changes in placental permeability from fetus to mother shown here might be more prominent in the human. The responses of these two species to an inflammatory event are similar with respect to the three plasma proteins AFP, transferrin and transthyretin (prealbumin); as in this study, these proteins have been reported to be acute phase negative proteins under inflammatory conditions (Heinrich et al., 1990; Hu et al., 2019; Mackiewicz et al., 1990). This supports the suggestion that these findings should be taken account of when advising pregnant women about the use of paracetamol. Given the unexpected findings of up-regulation of inflammatory cytokines and down-regulation of some acute phase plasma proteins, we are in the process of carrying out RNA-Seq replication studies and extending the range of cytokines estimated in fetal and maternal blood. Unfortunately, these experiments have been delayed by the COVID-19 emergency, which has closed our laboratories for an indefinite period. In view of the potential significance of our findings for the use of paracetamol in pregnancy, particularly the high frequency of its use, we feel it is justified to present these findings for peer review, in their present form.\n\nParacetamol (acetaminophen) is generally considered “safe” to use in pregnancy and lactation (Australian Medicines Handbook, 2019; Briggs et al., 2017) although it is one of the most commonly overdosed drugs, including in pregnancy (Rayburn et al., 1984). However, some authors urge caution in its use because of evidence of adverse effects (Brune et al., 2015). It has been reported that as many as nearly 80% of pregnant women in some populations ingest paracetamol (Dreyer et al., 2015). The findings of the present study, although based solely on experiments in rats, should be taken account of when advising pregnant patients on the use of paracetamol in pregnancy. The clinical situation is not straightforward because in addition to taking paracetamol to relieve pain, it may also be taken to reduce an increase in body temperature accompanying an infection (often respiratory), but there is evidence of an association between infection/fever and adverse outcomes for pregnancies; this seems to be a particular problem when infection/fever occurs at the beginning of the 3rd trimester (Hagberg et al., 2015). Thus, continued but limited use of paracetamol to control severe pain and to reduce body temperature at critical stages of pregnancy would seems to be appropriate but not the widespread use for lesser indications that is implied by the reports that most pregnant women take paracetamol.\n\nIncreased transfer of sucrose and AFP from fetal circulation into maternal circulation, as demonstrated in the present study, suggests that other molecules/metabolites could potentially also reach the maternal circulation. There are several clinical implications, including that increased AFP levels detected in pregnant women are used to detect potential neural tube closure defects, although this test is done earlier in pregnancy and we have as yet no evidence of paracetamol affecting placental permeability this early in pregnancy.\n\nFurther investigation is required to see if there are similar effects in the placentas of patients who have taken paracetamol. If the effect is indeed confined to the fetal side of the placenta it will be clinically difficult to determine such an effect in pregnant patients, particularly if it turns out to be variable as in our rat experiments, although transfer of AFP from fetal to maternal circulation might be a useful indicator.\n\n\nData availability\n\nRNA-Seq data on NCBI, Accession number PRJNA633629: https://identifiers.org/ncbi/bioproject:PRJNA633629\n\nFigshare: Effects of paracetamol on rat placenta and fatal brain. https://doi.org/10.26188/5ebff4c2781a0 (Koehn et al., 2020)\n\nThis project contains the following underlying data:\n\n- 200514 ELISA raw data.xlsx (raw data for the IL-1β ELISA )\n\n- 200514 sucrose permeability data.xlsx (brain, CSF and plasma levels of sucrose in pregnant rats and fetuses)\n\n- RA708 chronic high dose paracetamol.zip (plasma protein and H&E stained sections in Figure 10, A: RA708-50-04 HE x4.jpg, B: RA708-46-05 PP x4.jpg, C: RA708-46-05 PP x40.jpg\n\n- RA677 actute high dose paracetamol.zip (plasma protein stained section in Figure 10, D: RA677-41-03 x40 B.jpg)\n\n- 20191204 AFP loExp 1.tif (original unedited western blot image for Figure 8)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nRNA-Sequencing was undertaken by The Australian Genome Research Facility, Peter MacCallum Cancer Centre, Melbourne, Victoria.\n\n\nReferences\n\nAfgan E, Dannon Baker D, Batut B, et al.: The Galaxy Platform for Accessible, Reproducible and Collaborative Biomedical Analyses: 2018 Update. 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PubMed Abstract \| Publisher Full Text\n\nWyszynski DF, Shields KE: Frequency and type of medications and vaccines used during pregnancy. Obstet Med. 2016; 9(1): 21–27. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "64439", "date": "07 Jul 2020", "name": "Roland J. Bainton", "expertise": [ "Reviewer Expertise Blood brain barrier", "genomic toxicology", "biomarkers of stress and tissue injury", "blood diagnostics", "metabolic compensations of the CNS" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverview. The authors present a well-controlled immaculately-conceived and artfully interpreted paper on proteomic and genomic responses of the mammalian placenta to the most commonly administered drug in the world, paracetemol.\n\nThe results of this paper are quite profound and long overdue. There are few are very few studies that attempt as complete an evaluation of drug response and in an organ specific manner. This authorship team has exploited the most pertinent drug interfaces for chemoprotection of the fetus to glimpse the system of toxicologicologic protection of the developing fetus and brain. They exploit two well known pharmacologically highly regulated barrier interfaces: the placenta and BBB. As experts on barrier development they have the right expertise to measure the developmental role and robustness of these understudied barrier interfaces to the drug paracetemol. Tylenol (as known by the US brand name) is a ubiquitous pharmacologic agent used world-wide for the abrogation of pain and systemic suppression of inflammation. While deemed one of the safest medications ever invented because of its common utilization by every age group and gender, and its long standing well-described clinical toxicities suggests that it has been vetted for safety over and over. But with the right question and under the correct experimental circumstance profound novel sensitivities in the physiology of mammals can be discovered. Such is the insight of this manuscript.\n\nOf note. The paper is very complete. They demonstrate both acute and chronic changes to the placenta transcriptome with strong statistical relevance. Interestingly the chronic and acute genetic changes have few if any overlapping genes suggesting that long term toxicologic homeostasis may have very different effects to fetal development than single dosing. Thus, as noted by the authors, the use for the control of acute inflammatory responses may be warranted, but chronic ingestion any substance should be view with caution when the developing fetus is concerned.\n\nConclusion. This paper is well conceived, clearly written and expertly interpreted. Safety profiles of drugs are in flux and whether vertebrate homeostatic metabolic responses to drug exposure, acute or chronic, is truly benign is an open question. These authors clearly demonstrate, by the discovery of soluble protein changes in dosing of paracetamol, that there is more to learn about drug toxicology, in particular at the chemoprotective interfaces of the body, in this case the placenta and BBB. While the consequences of these proteomic changes are unclear they are corroborated by profound compensations in the transcriptional profiles of the placenta. Interestingly, the placental barrier does the lion share of compensation as the BBB of pups is nearly unchanged. This is a reassuring finding for the developing brain, but leaves many unanswered questions about how the fetus may affect maternal physiology (as noted by the authors).\n\nThe implications of this study are profound and not only for the use of paracetamol. In this paper they describe a road map for the study of all drugs that could have maternal fetal interactions and provide the physiologic and genomic insights to back up their assertions. Indeed their proposed experiments in pregnant women to follow up on their findings would be very important to the management of pregnancy and to the field of maternal/fetal physiology as a whole.\n\nI love this paper. BRAVO!\n\nMajor Issues None.\n\nMinor Issues. None.\n\nTypos. None.\n\nIs the work 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": "5793", "date": "19 Aug 2020", "name": "Norman Saunders", "role": "Author Response", "response": "We thank Dr Bainton for his detailed and perceptive commentary on our study of paracetamol/acetaminophen in pregnancy and in the newborn period. We particularly appreciate his view that our findings raise serious concerns about the use of this drug in pregnancy. At this stage we have only animal data that raises concerns, but we plan to follow up with human studies insofar as this is possible. We hope that our findings will give pause for thought by the regulatory authorities and doctors who regard paracetamol/acetaminophen as “safe” to be used in pregnancy and breast feeding, especially as the concept of “being safe” for any drug is a dubious one, and particularly for one that is used so frequently. We also appreciate the Reviewer’s comment that our approach provides a “roadmap” for studies of the many drugs that are prescribed in pregnancy about which there is little or no evidence on entry across the placenta and into the fetal brain. We are currently undertaking studies of psychotropic and anti-epileptic drugs as well new drugs introduced for the treatment of cystic fibrosis. Of course, the best outcome would be to find that little or no drug crosses the placenta and enters into the fetal brain. Dr Bainton’s comments are very important in helping to maintain this type of in vivo study." } ] }, { "id": "64436", "date": "20 Jul 2020", "name": "Patrizia Ferretti", "expertise": [ "Reviewer Expertise stem cells", "tissue repair", "developmental biology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper addresses an important and neglected question of potential negative effects of paracetamol during pregnancy. It examines gene expression changes in placenta and foetal brain and also presents out a number of functional studies to establish whether placenta permeability or brain barriers in the fetus are affected by the treatment. The study reports significant changes in the expression of a number of genes, including genes associated with the immune response, and validates changes in one of these genes, Il1ß, at the protein level in the placenta. While in some respects this study is still preliminary, the information presented here is valuable for underpinning future studies.\n\nThe authors clearly explain their choice of the end point (E19) selected, but not the reason for starting the chronic treatment at E15, rather than earlier, when important developmental events occur and teratogenic effects might be more likely and significant.\n\nSpecific Comments:\nP6: It is not clear why only the t-test was used when comparing multiple groups, as ANOVA followed by a post-hoc test should have been used.\n\nOn p7 the authors say “...65 up-regulated and 57 down-regulated....”, but Fig. 1 indicates 64 up-regulated genes, consistent wih the total of 121 up and down-regulated transcripts indicated in the right column.\n\nThe authors indicate that expression of 737 genes is significantly affected by chronic treatment, but do not show the level of significance. Does this mean that p is <0.05 (but never <0.01 or smaller) for all transcripts?\n\nTable 1 and 3. It would be helpful to colour code genes that change in both acute and chronic treatment groups and use thicker vertical lines between groups for ease of visualization.\n\nTable 2 includes genes that are not in the top 50 shown in Table 1, and this should be clearly stated (at a first glance the Table seemed a bit redundant). As for Table 1, the level of significance should be indicated. The Table could be made it easier to read if the “up-regulated (acute/control)” genes were shown below the “up-regulated (chronic/control)”, rather than in adjacent columns, or were clearly separated using a thicker vertical line. In addition, it is confusing to have a column “chronic/control” under the “up-regulated (acute/control)” list. This seems to have been done to accommodate S100a rather than inserting it under each comparison. Please check carefully that the difference indicated in different Tables are the same (e.g. S100a8 has a FC 2.25 in Tab1 and FC 2.26 in Table 2). “Il1b” should be changed to “Il1ß”. A pie chart of the inflammatory genes to complement Table 2 and Fig. 3 would be useful.\n\nP17, left column, top and Fig. 4. There is clearly variability, but to give numbers of fetuses where Il1ß levels could be detected over total numbers assayed for all groups would be more accurate and informative (e.g. acute 2/4, chronic low 7/16 and chronic high 10/19) than including these number only for the chronic high group, which appears to be wrongly given as 19/39, while the number of fetuses indicated for this group in Fig. 5 legend is 19.\n\nFigs 6 and 7 do not include error bars and no statistical analysis of these data seems to have been performed. It should be clearly indicated whether there was no statistical difference among groups at any time point studied.\n\nP20, left column, top. The statement: “AFP levels were higher in all treated dams compared to an un-treated control.” should be revised, as Fig. 8 shows an AFP increase only in chronically treated dams. It is a pity that the number of dams is too small to assess the significance of this observation and that no housekeeping protein was used to normalize AFP expression. If B is a densitometry of the gel in A, where according to the western blot labelling and the legend there is only 1 control for both dam and fetus, why are there 2 samples indicated in the controls in the charts? Given the low sample numbers and variability, particularly in fetal AFP levels, expressing the data as ratio is not appropriate.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5791", "date": "19 Aug 2020", "name": "Norman Saunders", "role": "Author Response", "response": "We should like to thank Professor Ferretti for her detailed review of this paper and in particular for her helpful suggestions for clarification and improvement of presentation of the results of this study. We provide responses below to all of the matters raised by Professor Ferretti. This paper addresses an important and neglected question of potential negative effects of paracetamol during pregnancy. It examines gene expression changes in placenta and foetal brain and also presents out a number of functional studies to establish whether placenta permeability or brain barriers in the fetus are affected by the treatment. The study reports significant changes in the expression of a number of genes, including genes associated with the immune response, and validates changes in one of these genes, Il1ß, at the protein level in the placenta. While in some respects this study is still preliminary, the information presented here is valuable for underpinning future studies. The authors clearly explain their choice of the end point (E19) selected, but not the reason for starting the chronic treatment at E15, rather than earlier, when important developmental events occur and teratogenic effects might be more likely and significant. The chronic treatment was limited to the last week of pregnancy in the rats, starting at E15, because it involved twice daily intraperitoneal (IP) injections of the drug and we were concerned that a longer treatment was an unreasonable imposition on the animals. In addition, it is well known that pregnant rats, if unduly stressed, are prone to aborting their fetuses. Also as mentioned in the 1st paragraph of the Discussion, the period of treatment covers about 25% of the gestational period of the rat, which is the main period that we are currently interested in. Oral medication would have been an alternative but it gives much less control over the amount of drug administered than when administered by IP injection. To have any control of oral administration requires monitoring of blood levels of a drug (itself an invasive process). This was not available at the time of this study but has now been developed for future studies. In relation to possible teratogenic effects, this was not an aim of the study and would, as the Reviewer indicates, require administration earlier in pregnancy. There is only limited information on possible teratogenic effects of paracetamol, probably because it came into clinical use long before specific regulatory requirements (e.g. FDA) for teratogenic testing in animals came into force. Studies involving limited epidemiological data have concluded that there is no evidence of an association between paracetamol ingestion and congenital malformations (Briggs et al. 2019, pp8-11). Specific Comments: P6: It is not clear why only the t-test was used when comparing multiple groups, as ANOVA followed by a post-hoc test should have been used. For the analysis of RNA-Seq data, the t-test is part of the packages we used and includes built in posthoc corrections for multiple comparisons. For the data on Il1ß the advice we have from our departmental statistical expert is that t-tests are appropriate for this type of research: (Lew M.J. (2019) A Reckless Guide to P-values. In: Bespalov A., Michel M., Steckler T. (eds) Good Research Practice in Non-Clinical Pharmacology and Biomedicine. Handbook of Experimental Pharmacology, vol 257. Springer, Cham.). Nevertheless in view of the Reviewer’s comment we have run the data through ANOVA followed by Tukey's posthoc test. The significance levels are the same as those we obtained with a t-test. We have added this information to the Methods subsection “Statistics”. On p7 the authors say “...65 up-regulated and 57 down-regulated....”, but Fig. 1 indicates 64 up-regulated genes, consistent with the total of 121 up and down-regulated transcripts indicated in the right column. We thank the Reviewer for drawing our attention to this error which has been corrected. The authors indicate that expression of 737 genes is significantly affected by chronic treatment, but do not show the level of significance. Does this mean that p is <0.05 (but never <0.01 or smaller) for all transcripts? As indicated in the Methods section on “Statistical Analysis” we used P<0.05 for two of the three analyses used. We focussed on genes with large fold changes as these are more likely to be of functional significance than would be indicated by a higher level of statistical significance. Table 1 and 3. It would be helpful to colour code genes that change in both acute and chronic treatment groups and use thicker vertical lines between groups for ease of visualization. We thank the Reviewer for this suggestion. The Tables have been modified accordingly. The treatment groups are now separated by a gap. The colour coding highlights some interesting differences in the number of genes that responded in the different treatment groups. A note of this has been added to the Table legends and in the text. Table 2 includes genes that are not in the top 50 shown in Table 1, and this should be clearly stated (at a first glance the Table seemed a bit redundant). This Table shows only inflammatory and immune-related genes and therefore some genes in the top 50 in Table 1 do not appear here. This is now indicated in the legend. As for Table 1, the level of significance should be indicated. P<0.05 added to legend. The Table could be made it easier to read if the “up-regulated (acute/control)” genes were shown below the “up-regulated (chronic/control)”, rather than in adjacent columns, or were clearly separated using a thicker vertical line. This change would make a very long 2 column table. We prefer the helpful suggestion that the columns should be separated which we have done with a narrow blank column In addition, it is confusing to have a column “chronic/control” under the “up-regulated (acute/control)” list. This seems to have been done to accommodate S100a rather than inserting it under each comparison. Unfortunately in the editorial process of preparing the pdf from the submitted Table spreadsheets some of the down-regulated genes have been sliced off and put incorrectly under the up-regulated categories. We are puzzled by this as the proof we received to check was correct. I have discussed this with the Editorial Office who have indicated that they will make sure this does not occur in the next version. Please check carefully that the difference indicated in different Tables are the same (e.g. S100a8 has a FC 2.25 in Table1 and FC 2.26 in Table 2). This was due to a difference in rounding, which has now been corrected. “Il1b” should be changed to “Il1ß”. Il1b is the notation used in the gene data base ncbi.nim.nih.gov, we would prefer to retain this notation in tables. A pie chart of the inflammatory genes to complement Table 2 and Fig. 3 would be useful. We generally find that pie charts are not helpful and would prefer not to make this addition. P17, left column, top and Fig. 4. There is clearly variability, but to give numbers of fetuses where Il1ß levels could be detected over total numbers assayed for all groups would be more accurate and informative (e.g. acute 2/4, chronic low 7/16 and chronic high 10/19) than including these number only for the chronic high group, which appears to be wrongly given as 19/39, while the number of fetuses indicated for this group in Fig. 5 legend is 19. We think that the Reviewer is probably referring to Fig 5. We agree that the way of representing these data that the Reviewer has suggested is clearer. This has been incorporated into the text (bottom P30). Figure 5 has been modified to make it clearer that values were obtained from 4 control fetuses. The legend has been re-written to make it clearer how many dams and fetuses were involved in this part of the study. Figs 6 and 7 do not include error bars and no statistical analysis of these data seems to have been performed. It should be clearly indicated whether there was no statistical difference among groups at any time point studied. Each point is a single fetus. The n values represent the number of fetuses in each treatment group. The legend has been rewritten to explain this more clearly. P20, left column, top. The statement: “AFP levels were higher in all treated dams compared to an un-treated control.” should be revised, as Fig. 8 shows an AFP increase only in chronically treated dams. This has been revised to state that there was an increase in dams’ AFP only in the chronically treated animals. It is a pity that the number of dams is too small to assess the significance of this observation and that no housekeeping protein was used to normalize AFP expression. If B is a densitometry of the gel in A, where according to the western blot labelling and the legend there is only 1 control for both dam and fetus, why are there 2 samples indicated in the controls in the charts? Given the low sample numbers and variability, particularly in fetal AFP levels, expressing the data as ratio is not appropriate. We agree that it is a pity that the numbers were very small, but we were constrained by the effects of being shut out of our laboratories for several months because of the coronavirus emergency. In general the only way to obtain accurate AFP values is to measure the actual concentrations of the protein. We are very aware that Western blots are only semi-quantitative at best. We attempted to make the gels from which we took measurements as comparable as possible within each age group by using similar volumes of plasma (or diluted sample). The concentrations of plasma proteins vary between different animals and are not related to each other therefore using albumin as a reference protein would not provide more clarity. We thank the reviewer for drawing our attention to the discrepancy in control adult numbers in the western blots (A) and in the densitometry readings (B). This has been corrected." } ] } ]	1	https://f1000research.com/articles/9-573
https://f1000research.com/articles/9-999/v1	18 Aug 20	{ "type": "Research Article", "title": "Discovery of potential immune epitopes and peptide vaccine design - a prophylactic strategy against Rift Valley fever virus", "authors": [ "Maruf Ahmed Bhuiyan", "Syeda Tasnim Quayum", "Foysal Ahammad", "Rahat Alam", "Abdus Samad", "Zulkar Nain", "Maruf Ahmed Bhuiyan", "Syeda Tasnim Quayum", "Foysal Ahammad", "Rahat Alam" ], "abstract": "Background: Rift Valley fever virus (RVFV) is an emerging arbovirus infecting both animals and humans. Any form of direct contact with body fluids, blood or tissue of infected animals is the mode of transmission of this pathogen. Despite being an emerging virus, no proper vaccinations are yet available for the public. Our objective is to compose a multiepitope vaccine utilizing immuno-bioinformatics as a strategy against RVFV. Methods: To identify immunodominant epitopes and design a potent vaccine candidate, we applied a series of immunoinformatic approaches with molecular dynamics and immune response simulation frameworks. Results: A glycoprotein with the highest antigenicity was selected and employed for determining promising epitopes. We selected T cell epitopes based on their immunological potencies and cytokine inducing properties, while B cell epitopes were selected based on their antigenic features. Finally, we selected four cytotoxic T-lymphocyte, two helper T-lymphocyte, and three linear B-lymphocyte epitopes that were arranged into a vaccine construct with appropriate adjuvants and linkers. The chimera protein was modeled, refined, and validated prior to docking against toll-like receptor 4. Docking studies suggest strong binding interactions while dynamics simulation revealed the stable nature of the docked complex. Furthermore, the immune simulation showed robust and prolonged immune responses with rapid antigen clearance. Finally, codon optimization and cloning conducted with Escherichia coli K12 suggests high translation efficiency within the host system. Conclusion: We believe that our designed multiepitope vaccine is a promising prophylactic candidate against RVFV pathogenesis.", "keywords": [ "Vaccine Design", "Immunoinformatics", "Rift Valley fever virus", "Immune Simulation" ], "content": "Abbreviations\n\n2D, two-dimensional; 3D, three-dimensional; ACC, auto cross co-variance; CAI, codon adaptation index; CTL, cytotoxic T-lymphocyte; E. coli, Escherichia coli; GC, guanine and cytosine; GRAVY, grand average of hydropathicity; HTL, helper T-lymphocyte; IEDB, immune epitope database; IFN, interferon; IFN-γ, interferon-gamma; IL, interleukin; IL-10, interleukin 10; IL-4, interleukin 4; kNN, k-nearest neighbors; LBL, linear B-lymphocyte; MHC, major histocompatibility complex; NCBI, National Center for Biotechnology Information; NMA, normal mode analysis; PDB, Protein Data Bank; PI, isoelectric point; RMSD, root mean square deviation; RVF, Rift Valley fever; RVFV, Rift Valley fever virus; SVM, support vector machine; TAP, transporter associated with antigen processing; TLR4, toll-like receptor 4.\n\n\nIntroduction\n\nRift Valley fever virus (RVFV) is a risk to worldwide public health and farming, especially in parts of Africa, Madagascar, and the Middle East1. RVFV epidemics have killed hundreds of thousands of animals, more than a thousand humans, and caused significant economic losses2. RVFV is a negative sense, single-stranded RNA (ssRNA) virus3 and belongs to the family of Bunyaviridae. RVFV was first identified in Kenya among sheep, in the vicinity of Lake Naivasha4. Using mosquitoes as a vector it can cause large scale transmission, causing mild symptoms like fever, back pain and nausea to fatal illnesses including critical eye diseases, encephalitis in humans, and lethal hemorrhage in animals5. Mortality of up to 90% has been reported in newborn animals and up to 30% in adult animals6, although the mortality rate for humans has been reported to be approximately 2%7. Epidemic alarms have persuaded several national and international health organizations to issue cautions about the rising risk of infection in Rift Valley fever (RVF) uninfected countries, like Europe and USA, due to the existence of vectors of transmission which are highly permissive, further compounded by global animal trade7–9. These reports unanimously concluded that coordinated efforts are needed in order to prepare for preventive measures against the recurrent emergence of RVFV.\n\nRVF is an arthropod-borne zoonotic infectious viral disease caused by RVFV9. Direct contact via tissue, body fluids or blood of RVFV infected animals acts as the prime mode of transmission for humans10. Mosquitoes are a major vector for RVFV spread11, infecting humans over long distances and even causing vertical transmission between livestock8. The incubation period for RVFV is about 2–6 days in humans. The virus consists of a negative sense, triple segmented (large, medium, and small) ssRNA molecule and has a viral genome encoding four proteins: glycoprotein, RNA-dependent RNA polymerase, non-structural protein, and nucleocapsid protein12–14. Although the non-structural proteins facilitate RVF to survive inside its host by inhibiting first-line immunogenic responses, its glycoproteins are essential and highly crucial for invasion, entry and viral replication inside the host cell13,14. Thus, the viral glycoproteins were targeted for our multiepitope vaccine design, which would be constructed using glycoprotein epitope sequences evoking an immune response inside the human system13,14. At present, epitope-based candidate vaccine design against viruses and bacteria as well as parasites has become very popular and has been done previously15–20. Multiepitope vaccines consist of short peptide fragments of immunogenic stimulants, which trigger a strong immune response and allow for a significantly lower chance of allergenic reactions inside the host system21. The identification of immunogenic epitopes derived from viral glycoprotein or nucleocapsid sequences has significantly enhanced the in silico development of peptide vaccines22.\n\nIn our study, we screened the RVFV proteome to find the highest antigenic glycoprotein to predict T and B cell epitopes using a computational approach. Subsequently, cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL) and linear B-lymphocyte (LBL) epitopes predicted to be immunogenic and antigenic were shortlisted, which were further subjected to toxicity and allergenicity analysis. A vaccine design was assembled by combining all the assessed CTL, HTL and LBL epitopes using linkers and an appropriate adjuvant. Physicochemical analysis and solubility prediction were performed in Escherichia coli (E. coli) to assess the vaccine. Next, secondary and tertiary vaccine models were predicted using structure analysis tools. The predicted tertiary structure was refined and validated. Moreover, a disulfide bond was introduced by disulfide engineering, improving vaccine stability. Interactions within the vaccine-TLR4 complex were studied using molecular docking and evaluated using molecular dynamics simulation. Additionally, codon optimization was carried out to increase the translation efficiency of the designed vaccine within a E. coli K12 host. Finally, immune simulation was carried out to predict real-life immunogenic potency of the vaccine. The employed steps for the development of vaccine are summarized in Figure 1.\n\nLBL, linear B-lymphocyte; HTL, helper T-lymphocyte; CTL, cytotoxic T-lymphocyte.\n\n\nMethods\n\nThe 232 complete proteomes of RVFV were obtained from ViPR (Virus Pathogen Resource) database (Supplementary Table 1, Extended data)23. ViPR is a reliable and open database for pathogenic virus families24. The viral proteome sequences retrieved were evaluated using VaxiJen v2.0 server for scores indicating antigenic influence. The threshold for antigenicity was fixed at 0.525. VaxiJen v2.0 sever possesses high prediction powers and utilizes auto cross-covariance (ACC) transformation methods. The glycoprotein sequence with the highest antigenicity score was taken from this proteome for further analysis.\n\nthe NetCTL v1.2 tool was utilized to isolate CTL peptides from the RVF glycoprotein sequence. This server generates different nonamer epitopes against 12 supertypes (A1, A2, A3, A24, A26, B7, B8, B27, B39, B44, B58, B62). NetCTL v1.2 identifies CTL epitopes depending on C-terminal cleavage, TAP competence and MHC-1 complex binding26. The threshold was determined at 0.5 with a corresponding sensitivity of 0.89 and specificity of 0.94. The MHC-I affinity for the CTL peptides was identified using the MHC-I IEDB web server27 and a consensus percentile of ≤5 was set to narrow down CTL epitopes, for obtaining high-affinity epitopes with MHC-1 alleles.\n\nThe isolated CTL epitopes were assessed for antigenicity by submitting them to the VaxiJen v2.0 server. To ensure proper induction of immune response in the human body, the immunogenicity of these epitopes was evaluated using the IEDB Immunogenicity tool28. Furthermore, AllerTOP v2.0 server was chosen for predicting allergenicity and ensure that the vaccine construct does not induce an allergic reaction in humans. AllerTOP v.2.0 uses k-nearest neighbors (kNN) methods to distinguish allergens from non-allergens29. Lastly, the selected nonamers were screened for probable toxicity using ToxinPred server, which predicts toxic epitopes using quantitative matrix methods and machine learning technology30.\n\nHTL acts as the orchestrator to stimulate B cells, macrophages, and CD8+ cells to fight against pathogens. Consequently, HTL epitopes are important for making an effective vaccine31. HTL epitopes, each 15-mer in length, were identified by utilizing the IEDB MHC-II tool. The anticipation of binding of the epitopes to class II alleles, HLA-DP, HLA-DR and HLA-DQ, were determined utilizing the consensus 2.22 prediction method on the same server32. Due to obtaining a large number of epitopes, a percentile rank of ≤0.3 was set as a threshold.\n\nHTLs release cytokines such as interferon-gamma (IFN-γ), interleukin 4 (IL-4), and interleukin 10 (IL-10) that can activate immune cells in the body. In addition, cytokines released by HTLs can survive past inflammatory responses and avert tissue damage. Therefore, HTL epitopes that can induce the release of cytokines are important in vaccine development. Therefore, in order to incorporate the epitopes that induce IFN- γ, we used the IFNepitope server, through the hybrid method (motif and SVM)33. Furthermore, the peptides were evaluated to check if they induce IL-4 and IL-10 using IL4pred and IL10pred servers, respectively34,35.\n\nThe activation of B cells plays a vital part in the activation of the humoral immune response and generation of plasma cells against a specific antigen, and distinguishing LBL epitopes is another vital step in the development of epitope-based vaccine constructs. LBL epitopes were predicted using a combinatorial algorithm of gradient boosting and a randomized tree method using the iBCE-EL server36. The predicted LBL epitopes were then reevaluated for their antigenicity score with VaxiJen v2.0 server, and non-allergenic and non-toxic epitopes were predicted using the AllerTOP v2.0 server and ToxinPred tool, respectively.\n\nFor an epitope to be antigenic and evoke a strong immune reaction it needs to be acknowledged by the MHC complex molecule. MHC alleles are the most polymorphic and occur in thousands of HLA combinations in humans. Therefore, an HLA allele with a high frequency of occurrences in the majority population of the world would have a high chance of exerting an effect immunogenic37. In our study, we wanted to find out the distribution, presence and frequency of the T cell epitopes, which were selected for the purpose of designing the vaccine structure. Allele Population Coverage of IEDB population coverage tool was utilized for the calculation of population coverage38. A prediction analysis was run on regions of Africa where the RVFV outbreak initially started and the neighboring countries that were mostly affected and also across the entire world.\n\nAll the predicted and assessed epitopes i.e. CTL, LBL, and HTL peptides were joined together using linkers and an adjuvant sequence was added upstream of all of them to form a vaccine structure. 50S ribosomal protein L7/L12 (GenPept Accession: P9WHE3) is a toll-like receptor 4 (TLR4) agonist and was chosen as an adjuvant for the construct to boost the immune response against it21. TLR4 can recognize viral glycoproteins and bacterial ligands39,40 stimulates the production of cytokines against them41. The CTL peptides and the 50S L7/L12 protein adjuvant were linked together with the EAAAK linker to ensure adequate separation between each component for their effective interaction with their respective targets. This linker was chosen as it separates bifunctional fused protein domains42. Each CTL epitope was separated using AAY linkers, and HTL epitopes by GPGPG linkers. Independent immunogenic potential of each LBL epitope was preserved by separating them using KK linkers43.\n\nTo avoid the risk of elicitation of autoimmunity by molecular mimicry, we screened the vaccine construct against the Homo sapiens protein sequences (NCBI: txid9606) through the use of NCBI BLASTp44.\n\nVaccines are developed to provide protection against diseases by evoking an immune response after injection against specific antigens, without causing any disease. Thus, vaccines need to have antigen inducing capability without being allergenic to the receiver. Therefore, the multiepitope vaccine construct was tested for antigenicity and allergenicity in each step of the initial design, using VaxiJen v2.025 and AllerTOP v2.0 servers, respectively29. Predictive assessment of different physiochemical attributes of the subunit vaccine was carried out using the ProtParam server45. Lastly, the SOLpro server in the SCRATCH suite was utilized to assess the solubility of the vaccine structure in E. coli, with a view to determining the bioavailability of the vaccine46.\n\nPredicted two dimensional (2D) configuration of our vaccine design was generated using the PSIPRED v4.0 web tool. The PSIPRED tool utilizes the query amino acid residues to predict a 2D model using two-feed-forward neural networking along with PSI-BLAST47. The three dimensional (3D) model of our designed vaccine was attained through the I-TASSER web tool, which exhibits protein modeling via a hierarchical procedure to estimate and bring about suitable structure and function48. The I-TASSER site helps to generate 3D structure of a protein and determine its functions using a state-of-the-art algorithm with high precision. This web tool enables determination of C-score, TM-score value and root mean square deviation (RMSD), along with the top five predicted structure models of the given protein sequence48. The produced 3D structure was chosen based on its C-score value and downloaded in PDB format. The server provides a C-score ranging between -5 to 2, where a higher value indicates a better model48.\n\nOur 3D vaccine structure was refined using the GalaxyRefine server. The web-based program reconstructs sidechains, then repacks them using dynamic simulations for proper structural relaxation49. The ProSA tool was used to recognize possible errors in the predicted tertiary model and for structural validation16. If the calculated score of ProSA deviates from the given range, this indicates that the primary sequence and predicted structure possibly contain flaws50. Additionally, the Verify3D server was employed to examine the congruity of a tertiary structure with its primary sequence. It is done by specifying a basic grade depending upon its position and surroundings and correlating the findings to known verified structures51. Ramachandran plot generation was carried out using the RAMPAGE tool52. The Ramachandran analysis plot is a visual representation of energetically permitted and rejected dihedral planar angles based on weak Van der Walls force of interactions between amino acids of the side chain. The RAMPAGE score enumerates the residues residing in favored, allowed and disallowed regions (in %) based on the PROCHECK principle53.\n\nDisulfide bonds are intra-protein bonds that can help stabilize tertiary or quaternary interactions in a protein54. Therefore, disulfide engineering was accomplished by introducing two novel bonds in the vaccine framework by employing the Disulfide by Design v2.12 web tool. The refined vaccine complex was uploaded, checked for a residue-pair match within parameters of χ3 angle -87° or +97° ± 30 and Cα-Cβ-Sγ angle 114.6° ± 10. Appropriate amino acid duos were chosen and mutated to cysteine residues55.\n\nCodon refinement is a vital step for the expression of the protein inside the host system. Therefore, codon adaptation was carried out by utilizing the JCat web tool in order to improve protein translation. E.coli K12 was selected for cloning since unadjusted codon sequences will reduce protein expression56. Termination of transcription (rho-independent), prokaryotic ribosome region of attachment and restriction enzyme cut-off sites were disregarded by setting the parameters to “avoid”. The server further provided two additional measures such as the codon adaptation index (CAI) and GC content of the refined sequences57. The codon nucleotide adapted sequence was cloned using SnapGene v5.0.8 software into a E. coli pET28a(+) vector obtained from SnapGene58. ApE and Genome Compiler are open access alternative to this software that could be used for this purpose.\n\nMolecular docking is a computerized process that evaluates and assesses the contact between a protein and its receptor. The binding affinity and interaction between the proteins provide a simulated score59. TLR4 can recognize viral glycoprotein60 and therefore, the TLR4 structure was obtained from PDB (PDB ID: 4G8A) and was chosen as a receptor to dock with the refined vaccine61. ClusPro v2.0 web docking server was appointed to determine the binding position and inclination for binding between the designed vaccine and TLR4 receptor62. The vaccine-receptor composite was selected based on the binding position (on active site), docking efficiency and the lowest energy scoring of the docked complexes.\n\nDetermining the stability and firmness of the protein-receptor docked structure by molecular dynamics is essential for carrying out an in silico study. Protein stability was resolved by contrasting protein dynamics and their counterpart normal modes63,64. the iMODS tool was used to evaluate and plot amino acid residues and their motion within their inner order via normal mode analysis (NMA)65. The NMA tool surfaced the extent of motion of the vaccine-receptor complex in terms of covariance and ability to deform; along with eigenvalues and B-factor results. The deformability of the complex depends on whether it can rotate each of its residues while staying in its position. The eigenvalue demonstrates the rigidity of motion, being directly proportional to the energy required for the deformation of the structure. Lower eigenvalues represent easier structural deformation65,66.\n\nSimulations of immune response of the vaccine design were executed with the C-IMMSIM v10.1 server. This server uses position-specific scoring matrix (PSSM) and AI-based technology to predict the intensity of immune reaction caused by the multiepitope vaccine67. It evaluates the immunologic response of the subunit vaccine according to an in vivo system and can also simulate immune response through its agent-based dynamic system. The minimum recommended interval is at least four weeks between the first and second dose68. Time-steps for each vaccine dose were fixed: 1, 84 and finally 168 respectively, with an interval of eight hours in-between. Analysis of three doses was conducted in respect to immune cell rise and activity with at least four weeks in between each of the dose sessions.\n\n\nResults\n\nAmino acid sequences of a total of 232 RVFV proteins were collected from the ViPR database. The VaxiJen v2.0 server revealed the two proteins with the highest antigenicity (Table 1). The glycoprotein sequence was selected for further analysis.\n\nNetCTL v1.2 server predicted 135 unique 9-mer CTL epitopes in total. Out of them, 27 epitopes were predicted as having the ability to act as both antigen and immunogen and were also nontoxic and nonallergenic (Supplementary Table 2, Extended data)23. Only six CTL peptides were shortlisted for the ultimate vaccine design based on a combined score (Table 2).\n\nSimilarly, 182 15-mer HTL epitopes and their MHC-II binding alleles were predicted and evaluated by utilizing the IEDB MHC-II tool. Next, cytokine production capability of the selected HTL epitopes was predicted for IL-10, IL-4 and IFN-γ. Only 14 epitopes were found to be capable of inducing any of the three cytokines’ production and to have antigenicity (Supplementary Table 3, Extended data)23. Of those that were capable of producing IFN-γ, two epitopes were chosen with the ability to produce IL-4 but not IL-10 and one epitope was chosen with the ability to produce IL-10 but not IL-4. Therefore, three epitopes were finally shortlisted prioritizing for IFN-γ and IL-4 production (Table 3).\n\nB cells act as antigen-presenting cells that recognize epitopes present on a protein and can bring about a humoral immune response69. The iBCE-EL sever program was used to predict unique LBL epitopes from the RVFV glycoprotein sequence. A total of 310 LBL epitopes were found. After further evaluation, 35 unique epitopes were found to be non-allergenic and non-toxic (Supplementary Table 4, Extended data)23. Amidst those, only five LBL epitopes were chosen for vaccine construction based on iBCE-EL server’s probability scores. LBL epitopes with higher probability scores were chosen. (Table 4).\n\n*C-score is the combined score provided by the NetCTL v1.2 server.\n\nIFN-γ, interferon gamma; IL-4, interleukin 4; IL-10, interleukin 10.\n\nThe CTL and HTL epitopes across African regions and Southwest Asia have a mean population coverage of 77.39 %, with an average hit of 1.78. The total combined class 1 and 2 epitopes have significant coverage across Europe (99.92%), North America (100%), South America (99.39%) and South Asia (99%), which indicates the multiepitope vaccine would be a good candidate in eliciting an immune response in these worldwide regions. In the African population, where the initial outbreak began, there is good coverage in the majority of regions, except for South Africa (18.36%), as shown in Figure 2.\n\nTo design our multiepitope vaccine, we picked four CTL epitopes (Table 2) based on their high immunogenicity and antigenicity scores and which were non-allergic and nontoxic. For the HTL epitope selection, we looked for epitopes that could produce all three types of cytokine; however, no epitope was found that had the capacity to induce all three types of cytokines (IFN-γ, IL-10, and IL-4). Hence, only two HTL epitopes were chosen from the glycoprotein sequence, which were capable of inducing IFN-γ and IL-4 (Table 3). Three LBL epitopes that were nontoxic and non-allergic and had the best iBCE-EL predicted probability, high antigenicity scores were selected (Table 4). Two extra epitopes were shortlisted for each of the three cases for the purpose of randomization to find the optimum vaccine construct. The choice of adjuvant was 50S ribosomal protein L7/L12, retrieved from the NCBI Protein database (Accession no. P9WHE3) and was linked to the selected epitopes using an EAAAK linker. For the final vaccine construct, four out of six CTL, two out of four HTL and three out of five LBL shortlisted epitopes were chosen based on different combinations and randomization to generate seven potential vaccine candidates, provided in Supplementary Table 5 (see Extended data)23, and were merged with AAY, GPGPG and KK linkers, respectively (Figure 3). The prospective vaccine with the ideal physicochemical property was chosen. The final assembled vaccine is 262 amino acid residues long.\n\nThe EAAAK linker was utilized to join the adjuvant at the 5’ end to the rest of the vaccine. CTL epitopes linked with AAY linkers, HTL epitopes linked with GPGPG linkers and form a bridge with the last CTL epitope and first HTL epitope. KK linkers were used to connect LBL epitopes and also form a bridge between the last HTL epitope and the first LBL epitope.\n\nLBL, linear B-lymphocyte; HTL, helper T-lymphocyte; CTL, cytotoxic T-lymphocyte.\n\nEvaluation of the multiepitope vaccine protein for similarity against the Homo sapiens proteome was done using BLASTp to ensure safety. No similar proteins were found, which negates the chance of autoimmunity as a result of the vaccine.\n\nPhysiochemical attributes of the vaccine structure were assessed using the ProtParam tool. The vaccine’s molecular weight was estimated to be ~28 kD and showed good antigenic characteristics. The predicted Isoelectric point (pI) of the designed vaccine is 4.96, indicating an acidic nature, and it has an instability index of 27.79, suggesting that the vaccine will maintain good stability inside the host system. The aliphatic index is 84.73, denoting the thermostable nature of the vaccine. The hydrophilic nature of the vaccine was estimated by the grand average of hydropathicity (GRAVY) score of -0.125. Half-life (t1/2) was calculated to be greater than 30 hours in a mammalian reticulocyte, greater than 20 hours in yeast, and more than 10 hours in E. coli. The overall vaccine construct is not allergenic and demonstrated good solubility as shown by the SOLpro server. The above-mentioned assessments suggest that our multiepitope vaccine design has the potential to be a good candidate for RVFV (Table 5).\n\nThe 2D structure of the vaccine design was acquired through the PSIPRED v4.0 workbench. The secondary structure consisted of 49.62% α-helices (130 amino acids), 11.83% β-strands (31 amino acids) and 38.55% random coils (101 amino acids) (Figure 4).\n\nHerein, α-helix, β-strands, and random coils are represented with pink, yellow and blue colors, respectively.\n\nThe multiepitope vaccine was speculated and constructed using I-TASSER server. The PDB structure (PDB ID: 1DD4) was regarded as the best template for modeling. This server carries out 3D modeling based on the consequence of threading template alignment and simulations are run by merging parameters of the structure assembly. It ranks the confidence of models quantitatively on C-score. The obtained C-score for the modeled 3D vaccine structure was -4.16. The refinement of the 3D structure was carried out by the GalaxyRefine web-tool. The refined and polished model was used to generate a Ramachandran plot, which showed an increased percentage of residues in the favored region. The refined vaccine construct showed 90.0% residues in the favorable region in Ramachandran plot. The global distance test (GDT-HA) score was found to be 0.9055, RMSD was 0.534, MolProbity was 2.209, clash score was 14.2 and poor rotamers were non-existent (Figure 5).\n\nThe refined tertiary structure was validated using the RAMPAGE server, ProSA-Web tool and Verify3D server. Validation carried out by the RAMPAGE server using a Ramachandran plot of the unrefined 3D structure showed 69.2% of residues in the favorable region, 20.8% in acceptable regions, and 10.0% of the residues in disallowed regions. After structural refinement was carried out, RAMPAGE revealed 90.4% of the vaccine’s residues resided in favorable regions, and 6.2% and 3.5% of residues were found in acceptable and disallowed regions, respectively (Figure 6A). The ProSA-web and Verify3D (Figure 7) servers were used to validate the unrefined tertiary structure. After refinement, ProSA-web revealed a Z-score of -6.2 for the best vaccine protein model, implying equivalence to the native protein conformation (Figure 6B).\n\n(A) Ramachandran plot analysis of the refined model showing favored, allowed and disallowed regions are 90.4%, 6.2% and 3.5%, respectively. (B) Validation using ProSA web tool, revealing a Z-score of -6.2.\n\nDisulfide bridging was used to stabilize the refined vaccine model. The introduction of cysteine residues was carried out via Disulfide by Design v2.12. Although 23 potential pairs of residues were found (Supplementary Table 7, Extended data)23 suitable for disulfide engineering, only two pairs of residues were chosen based on the energy of binding and χ3 angle. Accordingly, two mutations were created on a pair of selected amino acids, based on the lowest kcal/mol value. For, Leu67-Ala100 residues, the energy value was 2.40 kcal/mol and the χ3 angle was -103.13 degrees and for Phe28-Ala37 duos, the χ3 angle was -111.70 degrees and energy value was 2.86 kcal/mol (Figure 8).\n\nThe engineered pairs are indicated by blue and red colors.\n\nThe primary objective is to express our constructed multiepitope vaccine sequence using in silico cloning into the E. coli expression system. As such, the subunit vaccine construct requires codon optimization according to the codon usage for the expression pattern system in E. coli. To optimize for maximal expression of our vaccine design in E. coli K12, the JCat tool was used. The optimized codon sequence was 786 base pairs in length. The GC content was 49.75%, which lies within the ideal range of 30–70%. Codon Adaptation Index was 1.0, which is also within the ideal range (0.8–1.0). This ensures the efficient expression of the multiepitope vaccine inside E. coli. XhoI and BamHI restriction sites were inserted into E. coli plasmid pET28a(+) and the adapted vaccine’s codon sequences were inserted into a recombinant E. coli pET28a(+) plasmid using SnapGene software v5.0.8 (Figure 9). The final length of the recombinant construct is 6121 bp.\n\nOuter dark margin represents the vector skeleton.\n\nThe ClusPro v2.0 server tool was used to conduct molecular docking for characterizing contact between the illustrated refined vaccine and TLR4 (PDB ID: 4G8A). This server provided 30 different docked models, the best 10 of which were analyzed for the selection of the appropriate vaccine-receptor complex (Supplementary Table 6, Extended data)23. The model showing a strong interaction between vaccine residues and the active site of TLR4 along with the lowest energy value was selected. Model 7 fulfilled the above-mentioned criteria, and thus, was picked as the suitable vaccine-receptor complex (Figure 10). Model 7 had an energy score of -809.1 and indicated the highest binding affinity as it had the lowest energy score (Supplementary Table 6, Extended data)23.\n\nVaccine surface is represented in yellow, while the receptor is represented in cyan.\n\nTo understand the vaccine-receptor compound stability and its large-scale mobility, NMA was carried out using iMODS tool, which is simulated based on intramural configuration of the whole compound. Deformability of the vaccine-immune receptor complex depends on the isolated movement of every amino acid residue, illustrated via chain hinges (Figure 11). The eigenvalue was 2.443455e-557 (Figure 11A). The covariance matrix demonstrates the linkage between duplets of amino acids, the correlated residues marked in red, anti-related residues in white and non-correlated residues in blue, interspersed in dynamical zones (Figure 11B). The elastic mesh-work paradigm classifies which pairs of atoms are interlinked by springs and is visualized as a linking matrix. Dots are colored in accordance to their rigidity; the higher the rigidity, the darker the color (Figure 11C)70.\n\nMolecular dynamic simulation of vaccine-receptor complex, showing (A) eigenvalue; (B) covariance matrix; and (C) elastic network analysis.\n\nThe simulated immune response showed resemblance to an actual immune response against a pathogen (Figure 12). Subsequent exposures produced a higher tier immune response compared to the primary immune response. Secondary and tertiary responses showed higher levels of antibodies (i.e., IgM, IgG1, IgG2), which coincides with the waning of antigen (Figure 12A). This demonstrates the development of immune memory cells and, as a result, intensified antigen neutralization upon successive exposure (Figure 12A). Additionally, several B-lymphocyte cell isotypes with prolonged life have been noticed, signifying potential class switching and memory B cell generation (Figure 12B–12C). Increased responses were also observed in helper T cells and cytotoxic T cells with memory generation (Figure 12D–12F). An increase in macrophage activity and engagement was perceived, with a vigorous proliferation of dendritic cells (Figure 12G–12H). An increased amount of IFN-γ and IL-2 cytokines were also noticed (Figure 12l). These findings suggest the development of immune memory and, therefore, immunity against the virus.\n\nIn-silico immune response for vaccine construction as an antigen using C-IMMSIM server tool: (A) production of immunoglobulin due to injection of the vaccine; (B) B cell count following consecutive three injections; (C) population of B cell number per state; (D) helper T cell activation; (E) population of helper T cell number per state; (F) population of cytotoxic T cell number per state; (G) per state macrophage population; (H) dendritic cell population; and (I) level of cytokine and interleukins.\n\n\nDiscussion\n\nRVFV causes higher mortality and fatality among animals than in humans. Since this is a zoonotic virus, it can be transmitted through mosquitos and many regions like Africa, the Arabian Peninsula and their neighbors are at risk of an endemic outbreak7. Thus, a prophylactic measure against RVFV is essential. Vaccination is an ideal approach to gain immunity against infectious agents like RVFV. Development and manufacturing vaccines using a live or attenuated virus takes a huge amount of time and money. Moreover, higher antigenic presence in a weakened vaccine may overstimulate the protective immune system and complicate the situation by causing hypersensitivity reactions21. In comparison with conventional vaccine strategies, multiepitope vaccines possess no such complications71. Epitope-based vaccines could be a great choice in terms of safety, viability and economic rationality. In addition, potency as well as immune responses of multiepitope vaccines can be enhanced through the deliberate engineering of targeted epitopes72. Researchers have been looking for a way to reduce the budget for vaccine development, as well as minimizing allergenicity and side-effects of vaccines for quite a long time. With the emergence of new computational technology and widely available databases, different strategies are now available for designing and developing epitope-based vaccines following immunoinformatics expedients73,74. Vaccines against multitudes of viruses such as SARS-CoV-2, Ebola, Lassa, hepatitis C, Oropouche, Dengue and many more in the pipeline are prime examples of a structural vaccinology approach applied to the design of a multiepitope vaccine model17,19,75–79. In this study, our primary focus was to devise a multiepitope vaccine with the ability to produce robust immunity against RVFV having considered all the parameters of a subunit vaccine.\n\nAfter retrieving the 232 complete proteomes of RVFV from the ViPR server, screening for antigenic proteins was executed and glycoprotein sequences were selected based on higher antigenicity. A potent multiepitope vaccine should possess the potential to trigger B and T cell activation71. Therefore, potential CTL, HTL as well as LBL epitopic regions of RVFV glycoprotein were chosen for a candidate vaccine modeling. We were particularly interested in integrating B cell epitopes due to its function in antibody production and memory cell formation80. T cell-mediated immunity was also a concern in our vaccine design since plasma cells that give rise to humoral response reactions can easily be saturated by the deluge of antigens. Moreover, lifelong resistance can also be achieved by cell-mediated immunity or cytotoxic T cells81. Cytotoxic T cells can provide lifelong immunity through identifying and destroying infected cells82. Helper T cells, on the other hand, stimulate the release of IL-10, IL-4 and IFN-γ to overcome pro-inflammatory responses and lessen the damage caused to tissues and cells. Besides, they help to produce IgG antibodies and neutralize RVFV infection from peripheral system83. Furthermore, CD4+ cell activate B cells, macrophages, CD8+ cells when they come into contact with an antigen. Thus, all these points were considered and examined during our pursuit of designing the RVFV vaccine.\n\nIn a previous study, Adhikari and Rahman predicted overlapping immunodominant T cell epitopes from both nucleocapsid and glycoprotein84. However, their study concluded without formulating the vaccine candidate. Therefore, while Adhikari and Rahman focused on finding overlapping conserved CD8+ and CD4+ epitopes, our work was more centered on selecting CD8+ and CD4+ epitopes with high antigenicity, immunogenicity, non-allergenicity, non-toxicity and eventually tailoring them into a rational and potent vaccine candidate. Herein, unique epitopes from helper and cytotoxic T cells and B cells were chosen not only based on their antigenicity but also on other factors including allergenicity, immunogenicity and toxicity. The complete vaccine design was assembled by attaching the chosen CTL, followed by HTL and finally B cell peptides with the help of AAY, GPGPG, and KK linkers, respectively. Linkers were incorporated in vaccine construction as a part of an essential element to enhance stability, folding, and expression patterns of our vaccine protein85. The adjuvant L7/L12 ribosomal protein was attached to the first CTL epitope using the linker EAAAK. Multiepitope vaccines are less immunogenic when used alone due to a reduction in molecular weight compared to the protein, hence, it requires adjuvant to enhance its efficiency86. Adjuvants are components that help heighten cellular and humoral immunogenic responses for particular antigens as well as amplify the vaccine’s stability and longevity87,88.\n\nDue to the inclusion of immune dominant epitopes, the vaccine candidate was found to have higher antigenicity and immunogenicity while being devoid of allergenic feature. These are the prime features for a vaccine to be effective immune modulator in the first place. When it comes to peptide or subunit vaccines, the size of the vaccine candidate becomes an important matter. Interestingly, our designed multiepitope vaccine is only ~28 kD in size which makes it a suitable candidate vaccine. Solubility is another vital characteristic for any recombinant vaccine16. Luckily, our vaccine construct was predicted to be highly soluble inside the host E. coli system. Furthermore, physicochemical properties of the vaccine candidate were also in suitable range. For instance, the instability index suggested that the chimeric protein would remain stable after synthesis, while the GRAVY value and aliphatic index portrayed the vaccine to be hydrophilic and thermostable, respectively. However, the theoretical pI anticipated our vaccine as acidic which can be adjusted by modifying or adding some basic linkers or additional adjuvants.\n\nThe arrangement of crucial protein components was determined by 3D structure modeling, which was used for studying protein dynamics, functions, networking between residues and ligand interactions. The vaccine candidate that showed the best physio-chemical property was chosen and the vaccine construct was refined to significantly improve its expedient properties. The Ramachandran plot revealed 96.6% residues resided within the combined favored and acceptable regions along with only a couple of residues in disallowed region, which confirms the merit of the model. The GDT-HA and MolProbity score, along with clashscore, RMSD value and poor rotamer values were also indicative of sufficiency of the designed vaccine. The Z-score (-6.2) and Verify3D score (80.92%) also signal the satisfactory quality of the vaccine.\n\nMolecular docking as well as molecular dynamics simulation of the TLR4 and vaccine complex were performed in order to gain knowledge about the binding strength, contact nature and structural integrity. The vaccine-receptor complex underwent energy minimization, resulting in boosting of the stability of the total combination. The eigenvalue suggests the stiffness of motion of the molecules within the system and energy needed for their distortion. In our study, eigenvalue increases with each mode passed by which indicates more rigid and compact structure given the time.\n\nSerological evaluation to verify immuno-reactivity can be carried out for validating the candidate vaccine89. Thus, observation of the designed vaccine protein’s expression inside a suitable host is necessary. E. coli has been considered to be a great host choice for determining a recombinant protein’s expression patterns90,91. Hence, codon adaptation was accomplished for our recombinant vaccine to generate high levels of protein expression in E. coli K12. A CAI value of 1.0 and 49.75% GC content suggests the capability of optimum protein synthesis in the host. Moreover, two disulfide bridges were engineered into the vaccine for better stabilization, which is paramount for various biological and biotechnological applications. Animals immunized with glycoproteins of RVFV have been seen to produce protective antibodies against RVFV. Schmaljohn et al. have stated that mice inoculated with Sf9 cells expressing G1 and G2 glycoproteins of RVFV have produced antibodies that are adaptive in nature and neutralizes RVFV infection92. In a study conducted by Faburay et al. construction of a peptide vaccine consisting of two glycoprotein subunits Gn and Gc was carried out against RVFV, and injected into sheep to determine its neutralizing effects. Six sheep were immunized with their constructed subunit vaccine with a dose of 50 μg. The vaccine elicited a strong antibody response against RVFV which was confirmed using enzyme-linked immunosorbent assay (ELISA), suggesting recombinant glycoproteins can be used as a good subunit vaccine candidate. Faburay and his team further carried out a plaque reduction neutralization test to check the amount antibodies produced on primary and secondary dosages. Titer of first dose was low, but the second dosage increased the titer over 128014.\n\nIn current study, we also carried out the immune simulation of the vaccine candidate in an in silico immune simulator which demonstrated a good pattern of immune responses. As repetitive vaccine doses enhanced immune responses, we administered three doses that produced a variety of B-cell isotypes and T cell-mediated immune reactions with a noticeably significant number of memory B cells having a half-life of several months. Our simulated immune response shows a stronger and active immune response on secondary and tertiary doses, compared to the initial primary dose. IgG and IgM antibodies had an arbitrary titer of 80000 on second dose and 120000 on third dose. Sustained generation of IFN-γ and IL-2 was observed after multiple exposures of the vaccine as a result of increased helper T cell activation and therefore, the vaccine effectively stimulated a humoral immune response to ramp up immunoglobulin production.\n\n\nConclusion\n\nRVFV is a pathogenic agent with potential to become more widespread in upcoming times. Multiepitope vaccines provide a modular and tunable approach to vaccine design compared to traditional vaccine efforts. Our study focused on designing an effective and potent multiepitope vaccine to provide immunity against RVFV. The vaccine contained in silico assessed and predicted CTL, HTL, and LBL epitopes to produce an effective cellular and humoral immunologic response. Moreover, the vaccine simulation showed a potentially protective immune response and passed all molecular simulations with favorable results. However, further validation is required in in vivo and in vitro systems to guarantee the effectiveness of our designed vaccine.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nHarvard Dataverse: Extended DATA: Discovery of potential immune epitopes and peptide vaccine design - a prophylactic strategy against Rift Valley Fever Virus. https://doi.org/10.7910/DVN/B9Y1EH23.\n\nThis project contains the following extended data in DOCX format:\n\n- Supplementary Table 1 (All initially retrieved complete RVFV protein sequences)\n\n- Supplementary Table 2 (Predicted all potential cytotoxic T-lymphocyte epitopes)\n\n- Supplementary Table 3 (Predicted all potential helper T-lymphocyte epitopes)\n\n- Supplementary Table 4 (Predicted all potential linear B-lymphocyte epitopes)\n\n- Supplementary Table 5 (Physicochemical properties of all designed vaccine candidates)\n\n- Supplementary Table 6 (Scores of top-10 best vaccine-TLR4 docked complexes)\n\n- Supplementary Table 7 (Potential residue pairs for disulfide bridging)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\nMohammad Minnatul Karim confirms that the author has an appropriate level of expertise to conduct this research, and confirms that the submission is of an acceptable scientific standard. Mohammad Minnatul Karim declares they have no competing interests. Affiliation: Department of Biotechnology and Genetic Engineering, Islamic University, Bangladesh.", "appendix": "References\n\nDavies FG, Linthicum KJ, James AD: Rainfall and epizootic Rift Valley fever. Bull World Health Organ. 1985; 63(5): 941–3. PubMed Abstract \| Free Full Text\n\nBird BH, Ksiazek TG, Nichol ST, et al.: Rift Valley fever virus. J Am Vet Med Assoc. 2009; 234(7): 883–93. PubMed Abstract \| Publisher Full Text\n\nBouloy M, Weber F: Molecular Biology of Rift Valley Fever Virus. Open Virol J. 2010; 4(1): 8–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDaubney R, Hudson JR, Garnham PC: Enzootic hepatitis or rift valley fever. An undescribed virus disease of sheep cattle and man from east africa. J Pathol Bacteriol. 1931; 34(4): 545–79. Publisher Full Text\n\nIkegami T, Makino S: The Pathogenesis of Rift Valley Fever. Viruses. 2011; 3(5): 493–519. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "75911", "date": "14 Jan 2021", "name": "Firzan Nainu", "expertise": [ "Reviewer Expertise Bioinformatics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author appraised this paper by predicting epitope- and a peptide-based vaccine against glycoprotein of Rift Valley fever virus using immunoinformatics approaches. This manuscript has limited novelty, and most of the results presented by them are already published.\nHowever, your article is inadequately presented. Furthermore, there are many grammatical mistakes and spelling mistakes, as well.\nAlthough the article has scientific rigor, several major flows need to be corrected before indexing.\nThere are several limitations to this study listed in the comprehensive comments to the authors hereinafter. The presentation of findings and data interpretation in this manuscript could have been advanced compared to this existing design.\nMajor comments:\nGeneral Comments\nThe authors just have written several issues haphazardly. Many sentences/information throughout the manuscript have serious flaws that withdrawn my attention from it.\n\nMany non-scientific and incorrect/wrong information/sentences are there, which may mislead the readers.\n\nEvery section of the manuscript must be written scientifically according to the published literature with appropriate references.\n\nThe authors need to improve their writing style. The whole manuscript needs to undergo an English language edit.\n\nThe current manuscript will add no new information/recommendation for the readers.\n\nThe work seems preliminary. The study problem is not apparent.\n\nSpacing, punctuation marks, grammar, and spelling errors should be reviewed wholly.\n\nThe research questions could be defined more precisely. This is probably due to language concerns.\n\nIf the same work was performed on the SARS-CoV-2 (specific nCoV-2019), this would be wonderful.\nTitle:\nThe present title of the article is verbose. Authors need to shorten the title precisely for a clearer understanding.\n\nAlso, the current title indicates a consequence (e.g., immune epitopes and peptide vaccine design), which could be written as a statement instead.\nAbstract:\nThe purpose and significance of this research requisite to be explained in the abstract more clearly.\n\nThe abstract section is unsuitable—no focus point in the abstract section.\n\nChange the sentence “To identify immunodominant epitopes and design a potent vaccine candidate, we applied a series of immunoinformatic approaches with molecular dynamics and immune response simulation frameworks”. What does the series of immunoinformatic approaches mans?\n\nIncrease the sentences in the conclusion section.\nIntroduction:\nThe introduction section is inapplicable. Need to change the introduction considerably. Try to include the existing research limitations also, how the present research unravels those limits.\n\nThe study's gaps should be clearly defined in the introduction section with the applicable references.\n\nThe flow of the introduction is not perfect and unspecific. My advice is to make the sentences more lucid and legible for more productive comprehension.\n\nIn the introduction section, there are lots of redundant sentences repeating in the whole paper, making me feel the paper like a \"cut and paste\" from several other resources. For example, \"RVFV epidemics have killed hundreds of thousands of animals, more than a thousand humans, and caused significant economic losses.\" The sentence is not a way of scientific description. I suggest deleting sentences like these (there are more throughout the paper).\n\nThe paragraphs are not logically arranged; there are unnecessary repeats. Some of the explanation should be marked on their first appearance (for example, These reports unanimously concluded that coordinated efforts are needed to prepare for preventive measures against the recurrent emergence of RVFV), this makes me feel the authors didn't read through their paper after they finished it, and the abrupt terminologies make the paper like a Plagiarism.\n\nArrange the sentences (no meanings): \"Epidemic alarms have persuaded several national and international health organizations to issue cautions about the rising risk of infection in Rift Valley fever (RVF) uninfected countries, like Europe and USA, due to the existence of vectors of transmission which are highly permissive, further compounded by global animal trade.\"\n\nThere should be at least a brief discussion about peptide vaccines in epidemic situations in the introduction and references to any such vaccines proposed or underdevelopment for Rift Valley fever virus published materials, conference reports, etc.\nMaterials and Methods:\nIn silico prediction of immunogenic epitopes is unlikely fitting with the wet-lab data. Experimental validation of the predicted epitopes is highly required to recommend any predicted epitope as a potential vaccine.\n\nThe authors should need to add a workflow as a pictorial form, not the text form. It will help to comprehend easily for readers.\n\nMany of the tools used are not cutting-edge or represent the best available tools. For example, PSIPRED is not competitive in protein secondary structures. Authors are suggested to utilize the Rosetta or several other tools to predict protein 3D structures. The binding energy derived from it would be meaningful.\n\nSome tools used do not represent state of the art, and hence, the quality and confidence of the results may be limited.\n\nThe experimental section is poorly designed for \"Molecular docking between TLR4 and vaccine\". They could use them to analyze the active site instead of other servers that do not have proper validation or cross-checking with other tools.\n\nIn the method sections, the author's elaborate details regarding the software/website they have used—which need to describe briefly.\n\nTo get the hit or lead through virtual screening along with current finding, I strongly recommend to the authors to calculate the Binding Free Energy Calculation and molecular dynamics (MD) simulation (at least for 200 ns) to investigate the dynamic stability, the mechanism of binding, the behavior of structural conformation. These general strategies will support to get the best hit in terms of interaction, their stability, and to explore the mechanism of action. Authors also need to describe and illustrate how selectively their antigens will interact with the target to avoid nonspecific binding.\n\nNeed references for the following sentences \"Determining the stability and firmness of the protein-receptor docked structure by molecular dynamics is essential for carrying out an in silico study\" also for \"The NMA tool surfaced the extent of motion of the vaccine-receptor complex in terms of covariance and ability to deform; along with eigenvalues and B-factor results\".\n\nThe author here mentioned the identification of an active site by using a web server. However, the author needs to add some previous experimental data regarding the literature review to consolidate the viral proteome sequences by comparing them with the published articles.\n\nMajor weakness of the manuscript is the lack of controls (both positive and negative) for selected peptides/sequences. The authors only presented data for selected peptides, which makes score output hard to understand.\n\nA 1-2 sentence explanation of why some method was selected, how it works, and what the score means will make the manuscript easier to follow.\n\nFinally, the output of this work should be a list of potential epitopes to be tested experimentally. A pleasant addition would be a comparison with previously published findings.\n\nSeveral published works predict potential peptides for CTL, HTL, and LBL epitopes. It seems that those studies do not support the authors' proposal because NTKCRLSGTALIRAG, STAHEVVPF, or PNDYQSAHYLNN has not been nominated as a candidate peptide.\n\nThis is because it id hard to judge the algorithm adopted in the current study feasible or not.\n\nIt would add to the article's value if an MD (molecular dynamics at least 200ns) is included.\nResults:\nThe topic is interesting; however, the study is not solid enough to deserve indexing as it lacks any experimental proof. In fact, in the absence of any experimental work, this study represents just a hypothesis. The authors themselves keep repeating, \"this work needs experimental validation\"!\n\nResult section is poorly designed. Nothing was explained, although very little explanation can be found in the discussion section. Why do they want to design a peptide vaccine? The antigenicity and allergenicity evaluation of the vaccine construct they design lacks explanation.\n\nThe VaxiJen, which was developed over ten years ago, has been recently benchmarked against other methods (Dalsass et al., Front. Immunol. 2019). The recently published Vaxign-ML (Ong et al., Bioinformatics, 2020) work was reported to out-perform other prediction methods, including VaxiJen. The Vaxign-ML was extended to predict viral vaccine candidate prediction, specifically the whole SARS-CoV-2 proteome, and the SGP was predicted to have the highest score (Ong et al., bioRxiv, 2020). The authors should carefully compare their method and prediction results to the previous works (mainly focus on SARS-CoV-2; e.g., Abdelmageed et al., Biomed Res Int., 2020, etc.).\n\nI suggest the authors discuss the results obtained from the \"Estimated population coverage\" more in-depth.\n\nWhat is the need for \"Disulfide bridging in the vaccine structure\" and \"In silico immune responses\"?\n\nIt is not clear whether the current study's statistical methods are valid and correctively applied.\n\nIt is better to as a review to a bioinformatician, instead of molecular biologists.\n\nSome of the linear B cell epitopes might be too short to be considered. The authors could filter out epitopes based on length (e.g., seven amino acids).\n\nLimited novelty, and most of the results presented by them are already published.\nDiscussion:\n\nMany text repetitions found in the discussion section. It is highly recommended to emphasize findings and assumptions that support or disagree with other work(s).\n\nAddendum, repetition of the results should be avoided in the discussion section.\n\nA sound discussion includes principal, relationship, and generalizations supported by the results.\n\nThe discussion section should include a summary of why peptide/epitope vaccines are not licensed yet for human use.\n\nThe author should discuss more docking simulation studies in the discussion section.\n\nIn the docking part, more texts are needed regarding the interaction between the residues and the receptor.\nConclusions:\nNovelty of the work should be supplemented by the author (in the conclusion section).\n\nThis section should be supported by the results/insights. Conclusively, it will confer a distinct idea of the study.\n\nThe conclusion needs to address future perspectives.\nFigures:\n\nFigure legends are self-explanatory. But the repetition of the results and discussion should be avoided in the figure legends.\n\nThe resolution of figures can be improved.\n\nThe figures lack precision, units, and axis are not the well-label. The units are not standardized (and incorrect in some cases). Most of the figures do not have the proper resolution.\nReferences:\nFor better understanding, I feel this manuscript needs more detailed background information and precise explanation of their study in the Introduction and Discussion section.\n\nSeveral published articles related to SARS-CoV-2 must be included within the relevant text part of the manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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": "77290", "date": "28 Jan 2021", "name": "Sandra Moreno", "expertise": [ "Reviewer Expertise Virology", "Immune responses", "Vaccines", "Rift Valley fever virus" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, Maruf Ahmed Bhuiyan and colleagues use an exhaustive in silico approach to design a polypeptide sequence that includes an array of epitopes predicted that should theoretically induce both cell and antibody mediated immune responses in humans. In addition, they complete the study approach by adding predictions on populations immune responses based on HLA distributions, allergenicity responses, molecular docking to Toll like receptor 4, as well a simulation of immune responses upon vaccination. The authors conclude that this could be a promising vaccine candidate against RVF. As they mention, further investigations both in vitro and in vivo are warranted to ascertain its true potential to fight against RVFV. In our opinion the tools provided in this work constitutes an ideal in silico procedure. It remains however to be investigated further. For example, if this theoretical protein once expressed in a E coli system can be produced in sufficient amounts to be easily purified. On the other hand and, unfortunately, the article lacks novelty since it is a replication of other highly similar articles published by the same authors dealing with Lassa fever and, more recently, with SARS-CoV-2. Again and sadly, no data is yet available confirming that this methodology could help aiding vaccine design, since no in vivo experimental data has been yet provided. Not all the results are clearly presented, they make references to some computational scores and values that are not well explained. I would like to see some more references to other peptide vaccines for RVFV and other similar viruses. The study is very interesting but not as much as it could be if after modeling the vaccine, they had generated the vaccine and carried out in vivo tests. Some of their conclusions seem too categorical since all their data is based on informatics modeling (e.g. “there is good coverage in the majority of regions”, “These findings suggest the development of immune memory and, therefore, immunity against the virus”).To draw the proposed conclusions they should carry some experimental assays. Otherwise the authors should indicate the limitations of this approach.\n\nSpecific comments Abstract: Background:\n“Any form of direct contact with body fluids, blood or tissue of infected animals is the mode of transmission of this pathogen.” It should be included mosquito bites as a possible mode of transmission.\n\n“Despite being an emerging virus, no proper vaccinations are yet available for the public” . I would rather say no proper vaccines (rather that vaccinations) are available for human use or prophylaxis\nResults:\n“A glycoprotein with the highest antigenicity was selected. “Please explain whether Gn or Gc was selected. What is the criteria used to define high antigenicity?\n\n“We selected T cell epitopes based on their immunological potencies” What is the meaning for immunological potency?\n\n“The chimera protein was modelled, refined, and validated prior to docking against toll-like receptor 4.” Why against TLR-4? This is a membrane receptor and RVFV should be preferentially detected by endosomal TLRs. Explain if carbohydrate binding was considered for selecting TLR-4 or include references if available.\n\n“Finally, codon optimization and cloning conducted with Escherichia coli K12 suggests high translation efficiency” I do not understand this sentence. Would you mean that codon optimization was performed to increase translation efficiency?\nConclusion:\n“We believe that our designed multiepitope vaccine is a promising prophylactic candidate against RVFV pathogenesis.” This is a belief not supported by experimental in vivo testing. I would rather say this bioinformatics approach represents an strategy for rationally design of potential RVFV immunogens.\nIntroduction\n“RVF is an arthropod-borne zoonotic infectious viral disease caused by RVFV” Probably this sentence here is redundant.\n\n“…..and has a viral genome encoding four proteins:” This is only partially true. At least two additional proteins can be encoded by ribosomal frameshifts, including NSm and a 78kDa protein corresponding to NSm-Gn polypeptide. Please modify accordingly.\n\n“Although the non-structural proteins facilitate RVF to survive inside its host by inhibiting first-line immunogenic responses, its glycoproteins are essential and highly crucial for invasion, entry and viral replication inside the host cell” This is probably not accurate. Please rephrase. The role of glycoproteins in viral replication after entry has not been described.\n\n“…….to find the highest antigenic glycoprotein to predict T and B cell epitopes using a computational approach……” Should you mean “the highest antigenic glycoprotein T and B cell epitopes predicted using a computational approach”.\n\n“cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL) and linear B-lymphocyte (LBL) epitopes predicted” What is a linear B lymphocyte (LBL)? Should you mean “cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL) and B-lymphocyte (BL) linear epitopes predicted”\nMethods\n“TLR4 can recognize viral glycoprotein”. The provided reference does not support this claim. Revise for accuracy\nResults\n“The glycoprotein sequence was selected for further analysis.” Explain why the NSs protein was excluded from further analysis in spite of being more antigenic (table 1) and clarify which glycoprotein they refer to when selecting the different epitopes. (Note that RVFV encodes two glycoproteins upon posttranslational processing.)\n\nThe data shown in tables 2-4 is confusing. They shortlist different amount of epitopes in each table, refer to others in the text and then they only use some to assemble the vaccine construct.\n\nI think it would be useful to see more details in the inserted multiepitope in the map of plasmid construct (fig 9).\n\nThe units of the in silico immune response lack precision, sometimes units are not indicated. The antibody titers analyzed have a confusing scale and the immunization protocol is not clear. It would be interesting to know if these antibody levels could be related to a specific in vitro test.\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?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/9-999
https://f1000research.com/articles/9-997/v1	18 Aug 20	{ "type": "Research Article", "title": "Afrocentrism, national interest and citizen welfare in Nigeria’s foreign policy maneuvers", "authors": [ "George Chimdi Mbara", "Nirmala Gopal", "Nirmala Gopal" ], "abstract": "Background: Nigeria’s former Prime Minister, Sir Abubakar Tafawa Balewa, in his addresses of August and October 1, 1960, declared Africa as the centrepiece of Nigeria’s foreign policy. This policy thrust has remained a constant variable in the country’s diplomatic engagements over the years. The doctrine of Afrocentrism is predicated on the supposed manifest leadership role placed on Nigeria by nature. This made her leaders define Africa’s interest as Nigeria’s national interest, a development that has been contended to have no empirical bearing on the welfare of Nigerians thereby generating intense scrutiny. Consequently, this study evaluates the impact of Nigeria’s Afrocentric foreign policy thrust on the welfare of the ordinary Nigerians. The study further analyses the country’s gravitation towards citizen-centred diplomacy in 2007. These will help in comprehending the interaction between national interest and foreign policy in Nigeria, and to identify whose interests have been protected the most in Nigeria’s foreign policy pursuit – that of the ordinary citizens or the elites? Methods: Through the qualitative research method, in-depth interviews (IDIs) were conducted with Key Informants (KIs) for data collection. Responses from field study are merged with other primary and secondary sources of data to provide an incisive and balanced analysis that is premised on political realism. Results: Findings indicate that Nigeria’s international generosity and leadership role has never been predicated on a clear vision of national interest. Notwithstanding the flaws in Nigeria’s foreign policy over the years, this study also discovered that the outcome has not been a total failure as some respondents maintain. Conclusions: With the nation’s gravitation towards citizen-centred diplomacy, it is hoped that the country will put the interest of its citizens first in her policy pursuits.", "keywords": [ "Afrocentrism", "Citizen welfare", "Foreign policy", "National interest" ], "content": "Introduction\n\nFollowing the addresses by Nigeria’s former Prime Minister, Sir Abubakar Tafawa Balewa in August and on October 1, 1960, which pronounced Africa as the centrepiece of Nigeria’s foreign policy, this policy thrust has remained a constant variable in the country’s diplomatic engagements over the years (Adeniji, 2005; Akinterinwa, 2004; Dan-Fulani, 2014; Folarin, 2013; Jega, 2010; Saliu, 2006). Commenting on Afrocentrism, King (1996) cited in Folarin (2013), describes Africa-centred diplomacy as a political construct in which a country perceives the interests and welfare of the African region as critical to its interests and concerns as a nation. He describes it as an existential principle that sees a nation-state display a generous and magnanimous disposition towards African nations in need. Corroborating this view, Mazrui (2006) sees Nigeria’s Afrocentric policy as a Pan-Africanist worldview that has underscored its foreign policy since independence. By this disposition, Nigeria’s foreign policy gravitated around Africa and issues affecting the region received full attention ahead of matters outside the continent as exemplified in the formation of OAU and in solving the 1960–1965 Congolese crisis.\n\nIn her ‘rescue operations’, it is estimated that Nigeria has spent over 60 billion US dollars in financial assistance to countries in Africa and the Caribbean, not to mention the human cost (Fawole, 2003). No exact estimate has been made on the human cost of these operations. Its Africa-centred policy has been pursued at a very huge cost to the country and its people since independence. As early as 1960, Nigeria became actively involved in achieving international peace and security by contributing to global and regional peacekeeping, making her one of the highest contributors to United Nations peace operations. Among other operations, Nigeria supplied troops to DR Congo, Liberia, Sierra Leone, Somalia, Sudan, Sao Tome and Cote d’Ivoire.\n\nThe doctrine of Afrocentrism is predicated on the supposed manifest leadership role placed on Nigeria by nature, which made her leaders define Africa’s interest as Nigeria’s national interest (Warner, 2017); a development that has been contended to have no empirical bearing on the welfare of Nigerians thereby generating intense scrutiny (Dan-Fulani, 2014; Folarin, 2010; Mbara, 2019). In its Africa-first policy, did Nigeria play the felt leadership role and did other African states recognize and acknowledge the claim? Suffice it to say that it is one thing to see yourself as a leader, and it another thing to be accepted as one by others. Through the various military and civilian administrations since independence, this has remained a guiding principle of the nation’s policy constructs, applied at various degrees. To make matters worse, even with the country’s generosity, it is certain that her economic and social sacrifice has not yielded commensurate investments in human resource and capital development at home. Likewise, Nigeria’s “Big Brother” status has hardly been acknowledged, a clear indication that her claim to hegemony in Africa may have faded away. Besides that, the country’s respect and foreign image has been deteriorating as her citizens are constantly molested, harassed, unjustly detained and even killed abroad (Dan-Fulani, 2014; Fawole, 2003; Warner, 2017).\n\nConsequently, this study evaluates the impact of Nigeria’s Afrocentric foreign policy thrust on the welfare of the ordinary Nigerians. The study further analyses the country’s gravitation towards a citizen-centred diplomacy in 2007. These will help in comprehending the interaction between national interest and foreign policy in Nigeria, and to identify whose interests have been protected the most in Nigeria’s foreign policy pursuit – that of the ordinary citizens or the elites? Responses from field study are merged with other primary and secondary sources of data to provide an incisive and balanced analysis.\n\n\nMethods\n\nThe qualitative research method was used in this study. This research method involved in-depth Interviews (IDIs) with key informants (KIs) which were conducted for data collection. Through purposive sampling, the research population included relevant stakeholders in the country’s foreign policy formulation and implementation organs. Semi-structured in-depth interviews with “strategic informants” guided the data collection for the study as it relates to the various themes under investigation. Keen attention was paid to the responses of the participants to identify new areas of inquiry that are directly connected to the phenomenon under investigation.\n\nThis study took place in August and September of 2017 as a doctoral research thesis on “Nigeria’s Quest for a Permanent Seat on an Expanded UN Security Council: What Relevance for Domestic Factors?” The sample population was categorized into two sets of participants: the first includes a spokesperson from the Ministry of Foreign Affairs, and six senior officials/resource persons from the National Institute for Policy and Strategic Studies (NIPSS), Kuru, Plateau State, Nigeria. This first category of respondents is identified as “Group A.” The second category of respondents, identified as “Group B,” includes two academics and two postgraduate students from University of Jos who spoke on behalf of Nigerian students; one respondent from of the Federal Ministry of Information and Culture, and four “ordinary Nigerians” were carefully chosen from the petty traders, artisans and unemployed persons in the country to make up the sample population. These “ordinary Nigerians” represent the vox populi, they helped the researcher feel the pulse of the people on the street about the investigation. They were chosen through purposive sampling from recommendations of knowledgeable people and out of convenience for the researcher. The “ordinary Nigerians” were approached by the investigator in person. This brings the interviews conducted to a combined total of 16. This sample size was chosen in view of the principle of data saturation, that is, to avoid unnecessary repetition of data. These constitute the sample population for the primary source of data for the study. Each respondent was met at their respective workplaces for the interviews and each session lasted for about 25 to 35 minutes, depending on the flow of information. Interviews were recorded by the researcher using a smartphone and notes were taken during the sessions. The interview guides for each interview are available as Extended data (Mbara, 2020).\n\nThe data gathered were evaluated using content analysis, textual criticism and descriptive-historical analysis. The analyses were situated within the purview of the various research questions and in the light of the realist perspective on international politics. The study also utilised secondary sources of data archival materials, ranging from Nigerian government official documents, academic journals, newspapers, textbooks, conference papers, as well as reliable and verifiable internet materials.\n\n\nEthical issues\n\nEthical approval for this study was granted by the Humanities & Social Sciences Ethics Committee, University of KwaZulu-Natal, South Africa with protocol reference number: HSS/1033/017D. Prior to the collection of data for this study, gatekeeper’s permits were secured from the National Institute for Policy and Strategic Studies, Kuru and the Federal Ministry of Foreign Affairs, Abuja. For other respondents in the second category, an informed consent was received from each of them to participate in the study. In the informed consent form, respondents were assured that their views will be presented anonymously in the study, neither their names nor personal details were going to be disclosed while presenting the data. For this reason, respondents are represented with pseudonyms in this report and access to the interview recordings are restricted since they identify each respondent’s personal details. However, to replicate the study, an application can be made to the Humanities & Social Sciences Ethics Committee of the University of KwaZulu-Natal for access to the data.\n\n\nResults\n\nThe diplomacy of aid assistance in international relations has variously been explained (Dan-Fulani, 2014; Holsti, 1994; Mailafia, 2010). The realist perspective provides a concise explanation for the games nations play, namely, that countries offer aid to others based on calculated self-interest. Interests of this nature may be medium-term, or long-term, explicit, covert, or obscure. From this viewpoint, aid is one of the arsenals of economic diplomacy being deployed in the quest for national interest. Similarly, aid is also perceived as a form of imperialism. At the summit of the Cold War, when aid was used as part of the arsenals of ‘informal empire’, scholars like Hayter (1985) cited in Mailafia (2010), propagated the idea of “aid as imperialism,” a mechanism for courting friends as well as cajoling allies who were at the margins of the world capitalist system. On the other hand, states can and do offer aid for altruistic intentions. Wealthy nations come to the assistance of poorer neighbours for the purpose of charity and generosity. Likewise, in moments of a humanitarian crisis, conflict or natural disasters, most of this aid comes from an altruistic intention.\n\nNigeria has always seen itself as the regional hegemon in Africa, to the extent that the quest for Pax-Nigeriana has been a motivating factor, to varying degrees, of every Nigerian administration since independence, especially the regimes of 1960–1993 (Nuamah, 2003). To this end, Nigeria’s aspiration for continental leadership since independence is key to understanding some pivotal features of Nigeria’s foreign policy. Moreover, Pax-Nigeriana and Afrocentrism are structured around “concentric circles of foreign policy.”\n\nFour concentric circles underlie Nigeria’s grand strategy – from the innermost to the outermost. The innermost level deals with Nigeria’s relationship with its immediate neighbours (Sao Tome and Principe, Cameroon, Chad, Benin Republic, Niger and Equatorial Guinea). This is followed by Nigeria’s relations with other West African countries, Nigeria and the rest of Africa, then Nigeria in the world. (Respondent from the Federal Ministry of Foreign Affairs, Abuja.)\n\nAnalysts believe that Nigeria’s Afrocentrism was largely responsible for its dogged fight in dismantling apartheid (Adeniji, 2005; Amujiri et al., 2015; Dan-Fulani, 2014; Osuntokun, 2005). Nigeria internationalized the issue and made a huge financial commitment to fight the social/political menace. The robust economy of the 1970s won Nigeria the respect and credibility as the most populous black nation on earth and a credible voice to speak for Africa. This no doubt made the world to accept Nigeria’s proposal to establish anti-apartheid committees in both the OAU (now AU) and the UN with the country having permanent chairs on the committees (Gambari, 1997). Nigeria successfully got countries to boycott the 13th Commonwealth games and sanctioned countries and companies that continued to deal with the apartheid government (Ade-Ibijola, 2015; Amujiri et al., 2015; Hamil & Spemce, 1994).\n\nNigeria played an active role in the liberation of Southern Africa and the eradication of apartheid and colonialism, as well as supporting needy African countries with financial, material and technical aid over the years1 (Ade-Ibijola, 2014; Adeniji, 2005; Akpotor & Agbebaku, 2010; Dan-Fulani, 2014; Fawole, 2000; Kyenge, 2015). Similarly, Osuntokun (2005) commenting on the role Nigeria played in African decolonization efforts stated “[Nigeria] … sacrificed the goodwill of the West and economic development in order to see to the total liberation of Africa.” In the same vein, Garba (1987:101) asserts:\n\nNigeria… made enemies of erstwhile friends – all on account of their attitude towards the South Africa question. We have formulated economic policies that have sometimes been detrimental to our development because of our commitment to the eradication of apartheid.\n\nIn addition, Garba noted that the country lost the enormous sum of $45 billion over a period of 15 years for its embargo on exporting oil to apartheid South Africa. These facts bring us back to one of the research questions, what is the interaction between national interest and foreign policy in Nigeria, and does the interest represent the aspirations of its citizens? Going by the realist theoretical framework, which this study is built on, the obvious answer is No!\n\nNotwithstanding these successes, remarking on the Afrocentric policy, Amao & Uzodike (2015:10) averred:\n\nRegardless of these successes, this Africa-centred foreign policy concentration has not been without flaws. These flaws were soon to become evident in the downturn experienced by the country in its hitherto strong and viable economy and in the neglect of its domestic responsibilities, specifically the fulfilment of the social obligations expected of a government to its people. The resultant effect of this has been a steady decline in the nation’s oil revenue owing to a culture of poor maintenance, corruption and the extensive projects executed by Nigeria in other African countries.\n\nNigeria’s international generosity has never been predicated on a clear vision of national interest (Mailafia, 2010). The country’s interventionist role to achieve peace and security, protect democracy, offer grants, feed needy countries in the region and offer technical assistance has not yielded any noteworthy “dividend” to Nigeria or Nigerians in terms of investment opportunities from these benefitting countries nor has it enjoyed local support at home (Adeniji, 2005; Dan-Fulani, 2014; Folarin, 2013; Jega, 2010). The focusing of Nigeria’s foreign policy on Africa at independence has constituted a huge source of controversy to scholars, analyst and students of international politics. Some reckon that it is a noble course considering the reality and imperatives of the 1960s (Gambari, 1997; Garba, 1987). Notwithstanding, some scholars submit that it is a diplomatic blunder by a newly independent country whose leaders were unskilled in foreign policy articulation (Akinboye, 2013; Amao & Uzodike, 2015; Dan-Fulani, 2014; Fayomi et al., 2015; Mailafia, 2010). These scholars, in line with the realist outlook on international politics, believe that nation-building should have been accorded utmost priority through a partnership with developed countries to help the new state realize its full political and economic potential so as to become a haven for the black population all over the world. This is in accord with the realist standpoint which sees interest as the propelling force in international politics conceived in terms of power.\n\nScholars believe that excessive interference in the affairs of other countries and the decision to shoulder the collective burden of the whole of Africa made Nigeria miss opportunities to develop and grow domestically (Marafa, 2012). This policy thrust has been described as idealistic – hoping for a just, equitable and peaceful world. Thus, over the years, Nigeria ran its foreign policy like an international non-governmental organization (INGO), fostering negro-brotherhood and morality (Dan-Fulani, 2014). Afrocentrism ultimately drained funds that would have set the new nation on the right footing and propel the new country to greatness. Again, Dan-Fulani (2014) further observed that a realist approach that would guarantee substantial investments in science and technology, thereby moving the economy away from its agrarian nature would have been pursued and efforts made towards national unity. Besides, Nigeria’s alignment to the West, in practice, made it lose opportunities from the East which came with better conditions.\n\nFrom this realist viewpoint, it is regrettable that Nigeria has not deployed aid as one of its arsenals in economic diplomacy in its quest for national interest, neither has it deployed it as a tool for imperialism. In her membership and participation in regional organizations, Nigeria willingly shouldered, over and above, what was required of it. These extra burdens can be seen in the country’s statutory contributions to these regional groups, which she accepted, as commensurate with its perceived status. By way of example, Nigeria accounted for between 8 to 10% of the OAU’s regular budget. The same applies to its contribution to the African Development Bank (ADB) where she exerted herself “further by establishing a Trust Fund (NTF) in 1976 with an initial capital outlay of $80million. By December 1990, the NTF had financed 43 development projects in 17 sub-Saharan African states with a total value of $240.764 million” (Bukarambe, 2000:110). Nonetheless, in 1995, other African states voted against Nigeria’s candidate for the Presidency of the bank, a move that was engineered by the non-regional members of the bank. A source from the National Institute for Policy and Strategic Studies (NIPSS) (Group A) substantiates the above submission when he averred:\n\nAU is largely funded by 5 countries in Africa out of the 54 [member nations] … 5 countries contribute about 75% of the funding of AU, including Nigeria. But do you know that the other 4 have their citizens in strategic positions in AU except for Nigeria? So, how can you be spending such monumental resources and you cannot push your people further? Nigeria’s interest in this regard needs to be redefined. But then, the redefinition can only come when you have enlightened leadership. That is where the challenge lies (Respondent A, personal communication, September 21, 2017).\n\nAlthough the current President of the African Development Bank, Dr Akinwunmi Adesina, is a Nigerian, this is still a drop in the ocean.\n\nOn the country’s image problem, Warner (2017:9) contends, “Nigeria’s poor international reputation has led to a distinct dearth of soft power and resultantly, legitimacy problems, which it has assiduously sought to downplay or explain away”. The situation is so appalling that citizens of small neighbouring countries like Chad, Cameroon and Niger attack and harass communities along the borders with a great sense of impunity (Folarin, 2013). To underscore the importance of this point, Mailafia (2010:182) infers, “it is a paradox that while the country continues to expand its financial support to other countries, its image in the world continues to dwindle.” Nigeria’s image problem has so far defied all remedies as countless man-made and natural disasters continue to plague the country from all corners. Various factors lie at the centre of Nigeria’s lack of legitimacy in the international system. These include bad leadership, economic mismanagement, endemic corruption, ethno-religious violence and general perception, in some quarters, of Nigerians as arrogant, brash and loud (Adebajo, 2008; Warner, 2017). These, among other factors, have combined to diminish Nigeria’s prestige and weaken its influence in regional and global affairs. Mustapha (2008:52) captures the implication of this image problem concisely, “Nigeria’s national reputation or identity has a bearing on its foreign policy… Simply stated, Nigeria’s national reputation in the international arena as a country of alleged fraudsters and drug barons makes some of its national foreign policy objectives very difficult to attain.” This factor continuously stands in the country’s way in its search for global recognition and power.\n\nMoreover, Akinterinwa (2012) and Folarin (2013) both observe that Nigeria’s first enemies are those countries that have benefitted from her Africa first policy. Akinterinwa (2012) makes particular reference to Nigeria’s bid for the non-permanent seat of the UNSC in 2009 where Liberia, Sierra Leone and Togo, who were not candidates for the position, voted for themselves – a case of discarding their votes instead of casting it for Nigeria, their “Big Brother”. Other cases of ingratitude for Nigeria’s benevolence in her Africa first policy include Ghana (Nigeria supplies electricity on its behalf to Togo and Benin), South Africa (For whom Nigeria made enemies of erstwhile friends as it fought to liberate SA from apartheid), and Egypt (Nigeria mobilized support for it during the 1973 Yom Kippur war). These countries have been contesting for the proposed UNSC permanent seat against Nigeria’s ambition. Folarin (2013) adds that the so-called big powers in international politics today, use such soft economic and socio-cultural diplomacy to accentuate their indisputable hegemony rather than wasting resources on countries that will subsequently turn against them. Another respondent from NIPSS (Group A) makes the following submission on the level of ingratitude Nigeria experiences from its beneficiaries:\n\nNigeria has been playing a fatherly role in Africa, but what has Nigeria gained? Nothing! When the goodies are been shared, Nigeria is been relegated, but when the work is needed, Nigeria comes to do the work and at the end of the day what do we have to show for it? (Respondent C, personal communication, September 17, 2017).\n\nAs time went by, Nigeria’s mediatory role on the continent began to reduce and be taken for granted. For example, the 1976 issue between Kenya and Uganda (both members of the East Africa Community, EAC), where Kenya denied the landlocked Uganda access to its ports. Nigeria joined Uganda in pleading to Kenya to restore the latter’s access to the ports all to no avail. However, immediately after Henry Kissinger, America’s Secretary of State stepped into the conflict, Kenya relented and reopened the ports within 48 hours. Commenting on the development, Major General Joseph Nanven Garba, Nigeria’s External Affairs Minister at the time, observed that he was “amazed by the swiftness with which this promise was carried out; within 48 hours, the blockade had been lifted” (Bukarambe, 2000:111). A similar experience happened when Nigeria tried to mediate in the Ogaden War between Ethiopia and Somalia, and between Zaire and Angola over Shaba. In both cases, the disputing parties appeared to be receptive of Nigeria’s intervention but ended up resolving the issues by taking up arms against each other with the connivance and assistance of foreign powers. These are all clear indications that Nigeria’s perceived “Big Brother” status is hardly recognized and acknowledged by fellow African states. Why you may ask? The accepted linkage between domestic and foreign policy helps in explaining Nigeria’s failure to achieve the long-desired leadership role in Africa.\n\nScholars and respondents have been quite unanimous in adducing reasons for Nigeria’s waning regional influence – leadership failure, corruption, weak economic structures, ethnic and sectarian crisis, infrastructure decay and so on have made Nigeria an object of ridicule in the international circles, eclipsing her erstwhile appreciable image (Ade-Ibijola, 2015; Folarin, 2013; Jega, 2010; Mbara, 2019; Warner, 2017). These conditions have negatively constrained Nigeria’s foreign policymaking and implementation and reduced her claim of Pax-Nigeriana to an illusory hegemony in the region. Nigeria’s illusory hegemony has elicited a negative response from sister regional members instead of strengthening her claim to the status. Although Nigerian leaders and some Nigerians still believe in Nigeria’s leadership role in Africa, many scholars and observers think otherwise (Adeniji, 2005; Mbara, 2019; Saliu & Omotola 2008; Warner, 2017). Poverty, insecurity, tribalism and religious bigotry have affected the citizens’ support for their nation’s diplomatic engagements at the international scene and fundamentally constrained her claim to hegemonic status.\n\nThe following discussion is instructive given that power theory, which this study is built around, contends that state actors in the international system are principally motivated by national interest, which they feel morally obliged to pursue. In the case of Nigeria, however, the country’s pacifist foreign policy saw her lose the oil-rich Bakassi Peninsula to Cameroon in 2002 through the ruling of the International Court of Justice (ICJ), and Equatorial Guinea took Nigeria to task over territorial waters in the Atlantic among other known affronts against the “Giant of Africa”. Nigeria could have used its diplomatic weight within the continent to deter such insults (Folarin, 2013). Scholars agree that the fear of Nigeria has long ceased to be the first step to wisdom for many African countries who now take for granted Nigeria’s defeatist, pacifist and feeble approach to dealing with regional issues because of its self-proclaimed commitment to African brotherhood and good neighbourliness (Amao & Uzodike, 2015; Folarin, 2013; Mbara, 2019). The pertinent question is, where does the interest of Nigerian citizens feature in all of this? Which sane country cedes part of its territory and population to another country, not to mention a viable part for that matter? Was the ceding of Bakassi in the interest of the ordinary citizen or the interest of the political elites? Vox populi suggests that President Obasanjo ceded the territory to launder his image in the international community, a case where the interest of a leader becomes the interest of the people. This is the epitome of economic miscalculation and political rascality. Nigeria’s sovereignty has been undermined on all fronts.\n\nAdditionally, Mailafia (2010) and Dan-Fulani (2014) submit that national interest which is the guiding principle of the realist foreign policy has no doubt, been absent in Nigeria’s foreign policy endeavours since 1960. Dan-Fulani notes that the country’s membership of many bilateral and multilateral agreements at the sub-regional and regional levels have reduced the country to “a beast of burden” yoked with responsibilities that have no empirical bearing on the country’s national interest in this transient world. Furthermore, Nigeria, he notes is contributing more than its fair share in the sustenance of the African Union’s headquarters in the Ethiopian capital and has played a key part in the Unions specialized agencies and programmes not forgetting the enormous sacrifice it makes in peace operations around the world. If the AU headquarters was situated in Nigeria, perhaps it would have created jobs for the ordinary Nigerians thereby contributing to the economy and giving the country more relevance in global politics. Instead, such opportunities are being sponsored in another country.\n\nNigeria’s diplomatic activities in the international scene have been bereft of logic and national interest. For instance, Dan-Fulani (2014) describes the practice of maintaining full diplomatic missions in most African countries as a waste of economic and diplomatic resources. The same applies to the country’s membership of numerous international organizations that hardly has any benefit to the people2. Most of the missions on the continent have no strategic importance to the country, but only goes to serve her ego as the “Big Brother”, a status that has is hardly acknowledged by sister nations. To make matters worse, most of these countries do not have more than two dozen of Nigerians doing serious business in them and repatriating profits home neither is the mission serving any strategic interest bordering on our national security. In principle, Nigeria can make do with a maximum of twelve missions in Africa to achieve its goals in the region. Nigeria’s Consulate in South Africa can meaningfully manage the interest of the country in the entire Southern Africa Development Commission (SADC) axis; while the mission in Egypt will manage the country’s concerns in North Africa and parts of the Middle East. In the East African states, Nigerian High Commission in Uganda or Kenya can cater for the country’s interest in the sub-region. The country’s missions in immediate neighbours3 can be maintained for strategic and economic considerations. Around the West African sub-region, Nigeria’s missions in Ghana, Cote d’Ivoire and Senegal can be maintained for shared economic and strategic interests within the sub-region, as well as, geographical proximities (Dan-Fulani, 2014).\n\nAnother diplomatic endeavour that has not yielded commensurate gains for Nigeria is the country’s ECOWAS philosophy. Besides the general protocol regulating relationships, Nigeria is into several bilateral agreements with most countries in West Africa which have been lopsided. Nigeria solely financed the peacekeeping operations in Liberia and Sierra Leone and contributed 95% of the troops that executed the mission. To date, the precise amount spent on the mission has not been published – classified military information perhaps – but estimates show that millions of dollars (taxpayers’ money) were spent for this venture, which had no strategic nor economic interest to Nigeria. Strategically speaking, both Liberia and Sierra Leone have little geographical proximity with Nigeria as there are countries between them and the Atlantic Ocean covering thousands of miles in between. This reduces the probability of high flux of refugees or cross-border militia operations. In the area of economy, Nigeria’s military government at the time had no strategic plan on how to exploit the business opportunities presented by the intervention; there were no plans on how to take over mines and win contracts for the reconstruction efforts after the war. There were no plans for the post-war period which would serve Nigeria’s national interest (Dan-Fulani, 2014). Besides that, these missions lasted for years without UNSC recognition, financial or logistics support as it is the tradition with United Nations peacekeeping missions. Validating the above argument, another source from NIPSS avers:\n\n…You go to ECOWAS as a Nigerian, send in an application to go and work there. ECOWAS is 75% funded by Nigeria but the workforce comes from outside Nigeria. But in other climes, countries use their economic power to gain foreign recognition. When the US wanted Boutros-Ghali to leave as UN Secretary-General, it mainly withheld its annual contribution to the UN and the UN almost went bunkers, Boutros had to go. So, you cannot control such monumental economic influence, not to the advantage of your citizens. (Respondent D, personal communication, September 24, 2017)\n\nIn recent years, Ghana has exhibited hostile attitudes towards Nigeria and Nigerians living in the Republic. Policies to beat back Nigerian traders led the to the legislation that foreigners who wished to do business in the country must show a proof of 300,000 USD in its company account. This violates the ECOWAS protocol on free trade and market integration. These xenophobic attitude towards Nigeria and Nigerians was heightened on the night of 19 June 2020, when Nigeria’s High Commission in Ghana was demolished “under the full protection of State Security,” an act of naked aggression against the Nigerian state and in violation of Article 22 of the Vienna Convention on Diplomatic Relations (“Demolition of Nigerian High Commission,” 2020). Correspondingly, Nigeria has been supplying Niger Republic electricity (an essential commodity at home) since 1960 at a very subsidized rate. This is sequel to an agreement reached by the two countries in the 1960s, which prevented Niger from building a dam on the River Niger as it will obstruct the flow of water to the Kainji dam. Six decades later, it is time to review the agreement to reflect the current global economic realities.\n\nAgainst these backgrounds, there is a need to ask some critical questions: how has Nigeria’s foreign policy benefited the ordinary citizens of the country? Has the welfare and bare necessities of life of the common person been captured thus far? Most respondents strongly believe that foreign policy formulation and execution in Nigeria is an elitist affair. A respondent from group B maintains: When you talk of foreign policy, it has always been propagated by the elites. (Respondent B, personal communication, September 7, 2017). In the same way, respondent E maintains:\n\nNigeria’s foreign policy is more like deceit to the people in the sense that what Nigeria presents as foreign policy has never reflected on the people… See the case of Nigeria in Liberia and Sierra Leone in the late 1980s, [ECOMOG] See the way Nigerians are treated by these countries today… Let me be frank with you, it is total deceit [foreign policy]. Nigeria is a property owned by a few individuals (Personal communication, September 15, 2017).\n\nIn Nigeria (and most African countries), politics has become fierce and bloody, as it grants the winner control over state resources. This partly explains why those occupying state political positions cling to power so tenaciously that they even deny the losers their unalienable right. Under such circumstances, development as a process is absent from the manifesto of those in authority. Rather, their parochial interests are seen as development priorities of the people and foisted on them (Nuamah, 2003; Omoweh, 2000).\n\nThe elites have hijacked the state apparatus in Nigeria, thereby retarding the country’s quest for Pax-Nigeriana (Adebajo, 2008; Nuamah, 2003; Wright & Okolo, 1999). Analysing Nigeria’s foreign policy and domestic conditions, misdemeanours, Adebajo (2008:24) in his classic work, “Hegemony on a shoestring: Nigeria’s post-Cold War foreign policy” in Gulliver’s Troubles, compares Nigeria to a potentially wealthy Gulliver, and its leaders as the Lilliputians, “whose petty ambitions and often inhumane greed… have prevented a country of enormous potential from fulfilling its leadership aspirations and developmental potential.” Nigeria’s foreign policy averred Akinterinwa (2004) has never reflected the needs of the ordinary citizens; rather elitist considerations inspire its formulation, articulation and implementation. Thus, the needs of the cream of the society – the business class, bureaucrats, military and civil rulers are reflected in the country’s foreign policy. Corroborating this view, Adeniji (2004) submits that the common citizen in Nigeria has not been the focus of policy. The law, he notes, has been the focus, not the people. The people who made the law must always be placed above it in order of importance and defending a state whose citizens are worthless is equally useless. Likewise, Alh. Sule Lamido, Nigeria’s former Minister of Foreign Affairs observed that there is a disconnect between the public and foreign policy formulation process in Nigeria as “the people on the street” are left out in the decision-making process. Consequently, the Nigerian public has developed “policy apathy” towards government policies. It was for this reason, he notes, that the debt relief granted Nigeria in 2005 was not celebrated by the people on the street and many exhibit hostile tendencies towards Nigeria’s bid for a permanent seat on an expanded UN Security Council (Uhomoibhi, 2012). Corroborating this view, Warner (2017:10) asserts, “Nigerian civil society’s indifference toward the pursuit of Pax-Nigeriana could be at the heart of the country’s inability to achieve it.”\n\nIrrespective of the flaws in Nigeria’s foreign policy over the years, in fairness, this study also discovered that the outcome has not been a total failure as some respondents maintain. Citing Nigeria’s intervention in various crisis in West Africa (Liberia, Sierra Leone, Cote d’Ivoire, Mali and Gambia), respondent F from NIPSS submits that the benefits of these interventions:\n\n… Might not be clear at the immediate, but there is certainly something Nigeria has benefitted. By ensuring stability in the West African sub-region, Nigeria has also remained stable. When Liberia collapsed, more than 45% of the refugees came into Nigeria. However you look at it, it [the influx of refugees] had a negative impact on the Nigerian economy. In the mid-1970s and early 80s, when the Ghanaian economy crumbled, what was the next destination – Nigeria! … Virtually all Nigerian universities were taken over by Ghanaian scholars, some of them still here. Go to the University of Calabar, OAU [Obafemi Awolowo University] … Even if there appear to be no immediate gains for Nigeria when we look at it strategically. Nigeria has also succeeded in saving itself from the unnecessary influx of refugees. Look at the Great Lakes region in the Horn of Africa – DRC, Burundi, Rwanda, to some extent Kenya and Uganda. They have all been involved in one complex crisis threatening human existence. The country that has suffered it is Tanzania because of its stability. Tanzania has always at every point in time taken the chunk of the refugees that leave these countries. This has impacted significantly on the Tanzanian economy [See also the case of Zimbabwe and South Africa]. So, these are some of the things Nigeria may have benefitted [from its Africa first foreign policy]. But besides it, there is a need for Nigeria to redefine what its strategic interests are, especially as it has to do with its citizens in all its foreign engagements. It has to be clearly defined. Nigeria cannot be losing human and material resources of monumental magnitude only for its citizens to be treated with disdain in these countries. You cannot do that to the USA. If we are not seeking for spheres of interest or territories to occupy, we can at the international level gain some regard and respect for our citizens (Personal communication, September 18, 2017).\n\nThe Afrocentric policy without Nigerians is sterile. No matter how successful foreign policy is, without Nigerians as the immediate beneficiaries, it will likely not win the people’s support. To fill the gap, Uhomoibhi (2012) postulates a constructive and beneficial concentricism.\n\nIt was perhaps in response to these crucial questions and the image crisis the country was grappling with that Prof Dora Akunyili, Nigeria’s former Minister of Information and Culture, launched Nigeria’s rebranding campaign. Along with this came the need to reposition the country’s foreign policy. These two areas were given priority by the Umaru Musa Yar’Adua’s administration who believed that addressing the socio-economic and political problems in the country was a critical necessity. Moreover, Dan-Fulani (2014) observes that three major events in the international scene were also responsible for this move: the end of the Cold War, successful decolonization of Africa and the end of apartheid in South Africa made Afrocentrism an anachronistic idea in Nigeria’s foreign policy. The variables that necessitated this policy stand have given way to new challenges that require a complete repositioning of Nigeria’s foreign policy objective.\n\nLate Chief Ojo Maduekwe, Nigeria’s former Minister of Foreign Affairs during the Yar’Adua’s government, was credited for introducing this citizen diplomacy. Justifying its introduction, Maduekwe observed that as the largest concentration of Black people on earth, it behoved on the country to stand as a symbol of the black success story. Hence, citizen diplomacy suggests using foreign policy as a powerful weapon to showcase to the world who Nigeria and Nigerians are. It also implies that the global community must assume responsibility for its actions towards Nigerians in all circumstances (Maduekwe, 2007). Lending credence to the new diplomacy, Folarin (2013) supports this new policy position and Nigeria’s demand for respect from the international community by noting that the country’s large population, economic hegemony on the African continent and diplomatic track-record continentally and globally are enough credentials for it to be accorded the respect it demands and deserves.\n\nConversely, as laudable as this new citizen diplomacy may sound, scholars have been quite sceptical of its execution. Dickson (2010) cited in Amao & Uzodike (2015) observed that in 2007, a career diplomat from Nigeria, Dr Ngozi Ugo was nominated for the position of the Secretary General’s Deputy Special Representative and an Ombudsman at the UN. For her nomination to be confirmed, she required the diplomatic endorsement of her country’s government. However, owing to official bureaucracy and sheer incompetence from the Ministry of Foreign Affairs, she lost the position as time lapsed. What manner of citizen diplomacy is Nigeria practising when it cannot protect the interest of its citizens? Her presence would have boosted Nigeria’s quest for a UN permanent seat on a reformed Security Council and other agencies of the UN. Further, Dickson notes:\n\nOther more serious countries campaign for their citizens and that is why the highest-ranking African in the UN system is a Tanzanian woman. Go to the Commonwealth Secretariat in London you may think you are in India’s Ministry of Foreign Affairs because of the number of Indians there. And this is where our own Chief Anyaoku served for almost four decades. When is Nigeria going to stand and recognize its own? It is sad, unfortunate and indeed painful (cited in Amao & Uzodike, 2015:12).\n\nAdditionally, a series of examples abound where the Nigerian government has failed its citizens within the era of the new policy dispensation. Among them is the ceding of the oil rich Bakassi local government to Cameroon following the ruling of the ICJ. While the transfer of the territory is still in process, there has been massive reports of abuse, molestations and harassments from the Cameroonian authorities against Nigerians living in the area which is their ancestral home (Folarin, 2013:9). Others are the series of xenophobic attacks on Nigerians living in South Africa in 2008, 2015, 2017 and 2019.\n\nWhile scholars and foreign policy analysts believe that citizen diplomacy represents a radical shift away from the Africa-centred thrust, others suggest that the government may have only succeeded in achieving policy documentation rather than the actual execution. There is a need, therefore, for the government to be more proactive and responsive, both in words and deeds, to the predicaments of its citizens as the nation gravitates to the “people-first approach” diplomacy. Nigerians everywhere must-see sincerity in their government to protect and enhance their welfare.\n\n\nConclusion\n\nThis investigation made a retrospective look at Nigeria’s foreign policy in the last 55 years. The focus has remained constant – Africa and the senseless generosity to sister countries. Most supervising heads coin concepts that have no meaning or relevance in international relations and foreign policy articulation. As Nigeria pursued a busybody foreign policy, other countries with similar power base refused the temptation of extraterritorial activism within the region or elsewhere and chose to consolidate their power base for the greater good of their people. At independence, countries like Cote d’Ivoire and Senegal had a reasonably stable economy but chose to pursue foreign policies that would benefit their people through the Non-Aligned Movement (NAM). This study aligns with that of Jega (2010:7) who notes that despite significant external and domestic constraints, one sees much to be proud of as there are “notable and noteworthy accomplishments if only lessons could be learnt and reform initiatives launched appropriately.”\n\n\nData availability\n\nAccess to the interviews is restricted since they identify each respondent. However, researchers who wish to perform further analysis can make an application to the Humanities & Social Sciences Ethics Committee, University of KwaZulu-Natal, Westville Campus, Govan Mbeki Building, Private Bag X54001, Durban, 4001, South Africa for access to the data. Email: ximbap@ukzn.ac.za.\n\nFigshare: INTERVIEW SCHEDULES. https://doi.org/10.6084/m9.figshare.12609353.v2 (Mbara, 2020).\n\nThis project contains the questions asked to each group of participants during the key informant interviews.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Footnotes\n\n1 Nigeria, through its Technical Aid Corp (TAC) has over the years sent Scientists, technicians and medical practitioners to assist poorer and underdeveloped African countries by supplying them with free manpower to develop their industrial and science education.\n\n2 Nigeria belongs to 310 international organizations. Hence, the country pays over $70 million annually on levies and duties accruing to it from membership of such organizations. To avoid financial embarrassment, the country has decided to quit about 90 out of the 310 groups to save cost (Agbakwuru, 2017). It is instructive to note that most of these groups are duplicated or serve the same purpose as others.\n\n3 Benin Republic, Cameroon, Chad, Equatorial Guinea and Niger are Nigeria’s immediate neighbours.\n\n\nReferences\n\nAdebajo A: Hegemony on a shoestring: Nigeria’s post-Cold War foreign policy. In A. Adebajo & A.R. Mustapha (Eds.), Gulliver’s troubles: Nigeria’s foreign policy after the Cold War. Durban: University of KwaZulu-Natal. 2008; 1–37.\n\nAde-Ibijola AO: Nigeria’s ambition for the United Nations Security Council within the context of historical antecedents and domestic realities: Analysis of the prospects and challenges. Advances in Social Sciences Research Journal. 2015; 2(7). Publisher Full Text\n\nAde-Ibijola AO: Nigeria and the politics of African decolonization in the United Nations 1960-1994: Historical analysis and implications for Nigeria’s contemporary political ambitions. (Unpublished Doctoral Dissertation). University of Kwa-Zulu Natal, Pietermaritzburg, South Africa. 2014. Reference Source\n\nAdeniji A: Power and representation at the United Nations: A critique of Nigeria's bid for permanent seat in the Security Council. India Quarterly Journal. Indian Council of World Affairs, New Delhi. 2005; 61(2): 116–137. Publisher Full Text\n\nAdeniji O: New direction in Nigeria’s foreign policy. In B. A. Akinterinwa (Ed.), Nigeria’s new foreign policy thrust: Essays in honour of Ambassador Oluyemi Adeniji. Ibadan: Vantage Publishers Limited. 2004; 421–427.\n\nAgbakwuru J: Nigeria exits 90 int’l bodies to save image. Vanguard. 2017. Reference Source\n\nAkinboye SO: Beautiful abroad but ugly at home: Issues and contradictions in Nigeria’s foreign policy. Lagos: University of Lagos Inaugural Lecture Series. 2013.\n\nAkinterinwa BA: Concentricism in Nigeria’s foreign policy. In B.A. Akinterinwa (Ed.), Nigeria’s new foreign policy thrust: Essays in honour of Ambassador Oluyemi Adeniji. Ibadan: Vantage Publishers Limited. 2004; 428–460.\n\nAkinterinwa BA: Overview of Nigeria’s foreign policy, 1960–2010: Challenges and recommendations. In E. Anyaoku (Ed.), Review of Nigeria’s foreign policy: Issues and Challenges. Lagos: NIIA. 2012; 17–27.\n\nAkpotor AS, Agbebaku PE: The United Nations reforms and Nigeria’s quest for a permanent seat. J Soc Sci. 2010; 24(1): 51–55. Publisher Full Text\n\nAmao OB, Uzodike OU: Nigeria, Afro-centrism and conflict resolution: Five decades after - How far, how well? Afr Stud Q. 2015; 15(4). Reference Source\n\nAmujiri BA, AGU SU, Onodugo IC: Is Nigeria’s claim of leadership role in Africa a myth or reality? International Journal of Multidisciplinary Research and Development. 2015; 2(7): 343–353. Reference Source\n\nBukarambe B: Nigeria’s foreign policy in Africa, 1960 – 1999. In R.A. Akindele & B.E. Ate (Eds.), Selected readings on Nigeria’s foreign policy and international relations. NIIA enlightenment course series, Ibadan: Vantage Publishers. 2000; 1(1).\n\nDan-Fulani J: The end of apartheid: A redefinition of Nigeria foreign policy. IOSR Journal of Humanities and Social Science (IOSR-JHSS). 2014; 19(11): 53–57. Publisher Full Text\n\nDemolition of Nigerian High Commission Building in Accra. Guardian. 2020. Reference Source\n\nFawole A: Obasanjo’s foreign policy: Nigeria’s return to global reckoning? Nigerian Journal of International Affairs. 2000; 26(2). Reference Source\n\nFawole WA: Nigeria’s external relations and foreign policy under military rule, 1966-1999. Ile-Ife: Obafemi Awolowo University Press. 2003. Reference Source\n\nFayomi OO, Chidozie FC, Ajayi LA: Nigeria’s national image and her foreign policy: An exploratory approach. Open Journal of Political Science. 2015; 5(3): 180–196. Publisher Full Text\n\nFolarin S: Africa’s leadership challenges in the 21st century: A Nigerian perspective. Afr J Pol Sci Int Relat. 2010; 4(8). Reference Source\n\nFolarin S: Nigeria’s new “Citizen-Centred Diplomacy”: Any lessons from the United States? Fulbright American Studies Institute Fellow. Walker Institute of International and Area Studies WIIAS,. 440 Gambrell Hall, University of South Carolina, Columbia. 2013.\n\nGambari: Reform of the United Nations and Nigeria's quest for a permanent seat in the UN Security Council. Lagos: NIIA Founder's Day Lecture. 1997.\n\nGarba J: Diplomatic soldering: The conduct of Nigerian foreign policy, 1975-1979. Ibadan: Spectrum Books Ltd. 1987.\n\nHamil J, Spemce JE: South Africa's Watershed Election. The World Today. 1994; 50(7): 128–132. Reference Source\n\nHolsti KJ: International politics: A framework of analysis (6th ed.). Eaglewood, New Jersey: Prentice-Hall. 1994.\n\nJega AM: Nigeria’s foreign policy and the promotion of peace, development and democracy. In A.M. Jega & J.W. Farris (Eds.), Nigeria at fifty: Contributions to peace, democracy, and development. Abuja: Distributed for the Shehu Musa Yar’Adua Foundation, Lynne Reinner Publishers. 2010; 1–13. Reference Source\n\nKyenge TK: An appraisal of the role of the United Nations Security Council in the maintenance of international peace and security. (Unpublished Doctoral Dissertation) University of Jos, Jos, Nigeria. 2015.\n\nMaduekwe O: Time for a Citizen-Centred Diplomacy. 2007.\n\nMailafia O: Prometheus as Good Samaritan: Nigeria’s bilateral and multilateral assistance since independence. In A.M. Jega & J.W. Farris (Eds.), Nigeria at fifty: contributions to peace, democracy, and development. Abuja: Distributed for the Shehu Musa Yar’Adua Foundation, Lynne Reinner Publishers. 2010; 160–187.\n\nMarafa LM: Sustaining Nigeria’s Leadership Role in Africa. Business Day. 2012.\n\nMazrui A: Tale of two Africans: Nigeria and Africa as contrasting visions. London: Adonis & Abbey. 2006. Reference Source\n\nMbara GC: INTERVIEW SCHEDULES. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12609353.v2\n\nMbara GC: Through the eye of a needle: an examination of Nigeria’s quest for a permanent UNSC chair. J Afr Foreign Aff. 2019; 6(1): 139–164. Publisher Full Text\n\nMustapha AR: The three faces of Nigeria’s foreign policy: Nationhood, identity, and external relations. In A. Adebajo & A.R. Mustapha (Eds.), Gulliver’s Troubles: Nigeria’s Foreign Policy after the Cold War. Durban, South Africa: University of KwaZulu-Natal Press. 2008; 41–57.\n\nNuamah R: Nigeria’s foreign policy after the Cold War: Domestic, regional and external influences. Oxford: International Peace Academy. 2003. Reference Source\n\nOmoweh DA: Dynamics of Globalization: Impact on Nigeria and Africa. In R.A. Akindele & B.E. Ate (Eds.), Selected readings on Nigeria’s foreign policy and international relations, NIIA enlightenment course series. Ibadan: Vantage Publishers. 2000; 1(1): 45–57.\n\nOsuntokun A: Nigeria and the United Nations: Service deserves its rewards. In B. A. Akinterinwa (Ed.), Nigeria and the United Nations Security Council,. Ibadan, Nigeria: Vantage Publishers. 2005; 234–242.\n\nSaliu HA: Nigeria and the African seat on the Security Council: Problems and benefits. In H.A. Saliu (Ed.), Essays on contemporary Nigerian foreign policy,. Ibadan: Vantage Publishers Ltd. 2006; 1: 178–198.\n\nSaliu HA, Omotola JS: Can Nigeria get a UN Security Council seat? S Afr J Int Aff. 2008; 15: 75–85. Publisher Full Text\n\nUhomoibhi M: An overview of Nigerian foreign relations: A practitioner’s perspective. In E. Anyaoku (Ed.), Review of Nigeria’s foreign policy: Issues and challenges. Lagos, NIIA, 2012; 1.\n\nWarner J: Nigeria and “Illusory Hegemony” in foreign and security policymaking: Pax-Nigeriana and the challenges of Boko Haram. Foreign Policy Analysis. 2017; 13(3): 638–661. Publisher Full Text\n\nWright S, Okolo JE: Nigeria: Aspirations of regional power. In S. Wright (Ed.), African foreign policies. Westview Press. 1999; 118–32. Publisher Full Text" }	[ { "id": "84323", "date": "10 Jun 2021", "name": "Ismail Bello", "expertise": [ "Reviewer Expertise Nigerian Foreign Policy", "Multinational Corporations", "and Sustainable Development Goals." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 contributes to the discussion on Nigeria's Afrocentric foreign policy which has been in place since 1960. The paper further examines the impact of this policy on the welfare of the common man in Nigeria. As stated in various foreign policy textbooks, foreign policy of each states should be premised on National Interest, which is basically the interest of the state and its citizens. This paper submits that the afrocentric policy played a key role in liberation and anti-aparthied struggle in Southern Africa. Howerever, policy leaders have often neglect their first responsibility which is to provide for Nigerians and improve on citizens welfare. This perhaps influenced the adoption of citizen diplomacy by late president Umar Musa Yaradua. The paper concludes that Afrocentric foreign policy adopted by the Nigerian is more of senseless generosity. This conclusion is in line with my thoughts that the Afrocentric policy is a Father Christmas diplomacy which hasnt yielded much to the Nigerian state.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "6980", "date": "02 Aug 2021", "name": "George Chimdi Mbara", "role": "Author Response", "response": "Dear Prof Bello, Thank you so much for accepting to review this paper. Indeed, your constructive feedback is well received. I hope our policymakers will have the time to read through this article as it will help sharpen the choices they make while steering the ship of state. I look forward to collaborating with you some day. Sincerely, George." } ] }, { "id": "86788", "date": "02 Jul 2021", "name": "Stephen Osaherumwen Idahosa", "expertise": [ "Reviewer Expertise Regional security", "insurgency", "spread of instability in the Sahel and Lake Chad regions", "insecurity", "terrorism and foreign 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\nI wish to thank the editors of the “F1000Research” for the opportunity they accorded me, to read the article “Afrocentrism, national interest and citizen welfare in Nigeria’s foreign policy maneuvers”. I found this work an interesting paper which deals with a contemporary and important debate surrounding Afrocentrism - Africa as the centrepiece of Nigeria’s foreign policy. In particular, by focusing its attention on Nigeria’s generosity and developed an analysis through the lens of the realist theoretical framework - the author states that “Regardless of these successes, this Africa-centred foreign policy concentration has not been without flaws. From this realist viewpoint, it is regrettable that Nigeria has not deployed aid as one of its arsenals in economic diplomacy in its quest for national interest, neither has it deployed it as a tool for imperialism” and that “Nigeria’s bid for the non-permanent seat of the UNSC in 2009 where Liberia, Sierra Leone, and Togo, who were not candidates for the position, voted for themselves – a case of discarding their votes instead of casting it for Nigeria, their ‘Big Brother’. Other cases of ingratitude for Nigeria’s benevolence in her Africa first policy include Ghana (Nigeria supplies electricity on its behalf to Togo and Benin), South Africa (For whom Nigeria made enemies of erstwhile friends as it fought to liberate SA from apartheid), and Egypt (Nigeria mobilized support for it during the 1973 Yom Kippur war). These countries have been contesting for the proposed UNSC permanent seat against Nigeria’s ambition.” In the end “Afrocentrism ultimately drained funds that would have set the new nation on the right footing and propel the new country to greatness.” This is an interesting claim that pushes me to support this article.\nA few suggestions:\nThe work is clearly and accurately presented, however, concerning the literature, I think there are enormous books written by retired Ambassadors that could be useful.\n\nThe study design is appropriate and it does have academic merit and the data could be replicated. In a further study, I would suggest that the conclusion be expanded to reflect more on the result and possible recommendations.\n\nThe research possesses more strengths than weaknesses. However, areas such as the contemporary state of funding of the Foreign Service and the lack of appropriate capital for the vehicular instrument - MFA, to ensure the implementation of Nigeria's Foreign policy is either scantly written on or never written on at all. I think these areas should be explored as they play a huge and enormous role in the success and discharge of Nigeria's Foreign Policy.\n\nTherefore, suffice it to state that, the article has much potential to be indexed as it pays attention to an area that is significantly important in Nigeria’s foreign policy, its articulation, and implementation. The paper is well written, well organised, and developed. I recommend it for indexing in this current form. Congratulations.\n\nIs the work 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": "6981", "date": "02 Aug 2021", "name": "George Chimdi Mbara", "role": "Author Response", "response": "Dear Prof Idahosa, Thank you so much for accepting to review this paper. Indeed, your constructive feedback is well received and will be infused into future studies on the subject matter. I hope our policymakers will have the time to read through this article as it will help sharpen the choices they make while steering the ship of state. I look forward to collaborating with you. Sincerely, George." } ] } ]	1	https://f1000research.com/articles/9-997
https://f1000research.com/articles/9-648/v1	26 Jun 20	{ "type": "Brief Report", "title": "An ice-binding protein from an Arctic grass, Leymus mollis", "authors": [ "Todd L. Sformo", "James A. Raymond", "Todd L. Sformo" ], "abstract": "Several cold-hardy grasses have been shown to have ice-binding proteins (IBPs) that protect against freeze-thaw injury. Here, we looked for IBP activity in an Alaskan coastal grass that had not previously been examined, Leymus mollis (Pooidae). Rhizome tissue had strong ice-structuring and ice recrystallization inhibiting (IRI) activities, indicating the probable presence of IBPs. The gene sequence of an IBP was obtained. The sequence encoded a 118-amino acid IRI domain that contained eight repeats. A 3D structure of the IRI domain was predicted from the structure of the IRI domain of the perennial ryegrass Lolium perenne. The predicted structure appeared to have the same eight beta-roll coils found in the L. perenne IBP.", "keywords": [ "Leymus mollis", "ice-binding protein", "Arctic", "Alaska", "Pooidae", "dune grass", "Beaufort Sea" ], "content": "Introduction\n\nWithin the grass family (Poaceae), the subfamily Pooideae includes many cold-adapted grasses including wheat, barley and forage grasses. These grasses have developed a large family of ice-binding proteins (IBPs) that protect the plants from freezing damage (Sandve et al., 2008). The IBPs, which have negligible effect on the freezing point, are characterized by a C-terminal domain consisting of several repeating units that is associated with strong ice recrystallization inhibition (IRI) activity (Sandve et al., 2008). This region is thus called the IRI domain. Ice recrystallization, in which larger ice grains grow at the expense of smaller grains, occurs mostly at warmer sub-zero temperatures and is thought to cause damage to cell walls. Two of the most studied of these IBPs are from the grasses Lolium perenne (Sidebottom et al., 2000) and Deschampsia antarctica (John et al., 2009). Their amino acid sequences are very similar in both the N-terminal and IRI domains. The 118-a.a. IRI domain of the L. perenne IBP has been crystallized and its structure determined by X-ray crystallography (Middleton et al., 2012). The IRI domain has a beta-roll fold with eight similar coils, one side of which is the predicted ice-binding site. The spacing of the coils is very close to the repeat distance along the a-axis of ice. Interestingly, heterologous expression of the Lolium IRI domain in tomato was also shown to increase chilling (4°C) tolerance (Balamurugan et al., 2018), although the mechanism remains unclear. Here, we describe a related IBP from a pooid grass from Utqiaġvik (Barrow), Alaska, Leymus mollis. This site, at 71.3° N, is the highest latitude at which IBPs have been examined in a grass and is 9° higher in latitude than the site where D. antarctica was sampled in the southern hemisphere.\n\nL. mollis, also known as American dune grass, is found in coastal habitats, especially sand dunes across North America and Greenland. It is typically subjected to many stresses, such as low nutrient levels, salty sand, salt spray, little freshwater, inundation during storms, wind abrasion, and ice storms (Gagné & Houle, 2002). L. mollis’s IBP sequence is very similar to the Lolium and Deschampsia sequences, but closest to wheat (Triticum) IBPs. We also show that an extract of the grass has strong ice-shaping and recrystallization inhibition properties and predict its structure based on the structure of L. perenne IBP.\n\n\nMethods\n\nA grass sample was collected from a gravel beach at Utqiaġvik (Barrow), Alaska (71.3°N) on 6 October 2019, and stored at -80°C. The grass was shipped frozen to University of Nevada Las Vegas for analysis, but accidentally rose to ~10°C for about one day during shipment.\n\nTo obtain a sample for measuring ice structuring and IRI activities, the brown outer layers of a rhizome from just below the soil surface were peeled off, revealing green tissue underneath. About 70 mg of the green tissue was ground in 1 ml water in a mortar and pestle. The suspension was centrifuged at 14,000 rpm for 5 minutes to yield a slightly cloudy supernatant. Stem tissue from lawn grass (Festuca sp.) at the University of Nevada Las Vegas (52 mg) was similarly homogenized and used as a control. Ice-structuring activity was observed by examining the growth of an ice seed crystal with well-defined ice c- and a-axes submerged in the grass extract supernatant, as described previously (Raymond & Fritsen, 2001). Briefly, the sample was placed in a rectangular tube and the tube was submerged in a controlled temperature bath with front and rear windows. The growth of the crystal was observed at a temperature slightly below the freezing point (~-0.2°C) with a horizontally mounted dissecting microscope. Ice-structuring activity was defined as the appearance of sharply defined facets on the crystal surface. IRI activity was observed as described previously (Raymond & Fritsen, 2001). Briefly, 3 µl drops of supernatant were placed on a slide cover glass. A liquid nitrogen-cooled slide glued to a handle was pressed against the drops to form highly polycrystalline ice between the slide and cover glass. The samples were stored at -3°C in hexane and changes in recrystallization were monitored in the temperature bath described above over 24 hours. Photographs were taken through crossed polarizers.\n\nGreen tissue was obtained and homogenized as described above. DNA was extracted with a NucleoSpin Plant II kit (Machery Nagel; catalog number 740770) according to the manufacturer’s instructions. To make IBP primers, the nucleotide sequences of IBPs from L. perenne, D. antarctica and Triticum aestivum (GenBank accession numbers EU680848, FJ663044 and KU204387, respectively) were aligned. Regions with high identities at the 5’ and 3’ ends were selected for primer sequences to obtain an amplicon that covered as much of the gene as possible. Primers for 18S ribosomal RNA were selected from conserved regions in the 18S sequences from Dupontia fisheri, L. perenne and D. antarctica (GenBank accession numbers KP794861, KJ598999 and MH628292, respectively) that flanked a region of high variability. At that time, we suspected the Utqiaġvik grass sample was D. fisheri and didn’t use the L. mollis 18S sequence. PCR was carried out with an Eppendorf Mastercycler Personal thermal cycler, with 3 minutes initial denaturation at 95°C, followed by 35 cycles of 30 seconds denaturation at 95°C, 30 seconds annealing at 59°C and 40 seconds extension at 72°C, followed by 3 minutes final extension at 72°C using Promega GoTaq polymerase (catalog number M300B). PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide and observed and photographed with a UVP transilluminator. IBP and 18S bands of the expected sizes were obtained (see Underlying data (Raymond, 2020c)), cleaned up with a Nucleospin gel and PCR clean-up kit (Machery Nagel, catalog number 740609) and sequenced in both directions at the UNLV Genomics Core with an Applied Biosystems 3130 sequencer. The primers that were used for sequencing are shown in Table 1.\n\nIBP, ice-binding protein.\n\nA 3D model of the IRI domain of the Leymus IBP was predicted with SWISS-MODEL (Waterhouse et al., 2018) using the eight-coil structure of the Lolium perenne IRI domain (Protein Data Bank accession no. 3ULT) (Middleton et al., 2012) as template. SWISS MODEL proposed three structures of the Leymus IRI domain based on the template. We selected the one with the highest sequence identity (76.5%), which also was the only one that had eight orderly coils like those in the L. perenne template. The free energy of this structure was then minimized (from -42.56 to -52.58 MJ mol-1) with the YASARA Energy Minimization Server (Krieger et al., 2009) and displayed with YASARA View. Stereoviews were obtained by rotating the molecule around its vertical axis by 3°. The average distance between coils in the IRI domain was measured with the YASARA distance function as the distance between the alpha carbons of Ser15 and Ser116 divided by seven.\n\n\nResults and discussion\n\nThe obtained 18S rRNA sequence (GenBank accession no. MT506010) most closely matched the sequence of Leymus mollis (GenBank accession no. EF581964) (98.3% identity). Among the Pooidae, L. mollis is a member of the Triticodae, which includes wheat (Triticum) and barley (Hordeum), while Lolium and Deschampsia are members of the Poodae. The grass was confirmed as L. mollis from photos of the remnant spikes and leaves by Matthew Carlson (University of Alaska Anchorage) and Carolyn Parker, University of Alaska Fairbanks (personal communications to T.S.).\n\nAn extract from green rhizome tissue strongly affected the growth of an ice seed crystal, causing it to develop sharp facets (Figure 1A). The facets are an indication of the presence of ice-binding proteins. Extract from lawn grass stem tissue (control) showed no such activity (Figure 1B). The Leymus extract also had strong IRI activity. A polycrystalline ice crystal recrystallized significantly after 21 hours at -3°C, while the supernatant of the grass extract showed no recrystallization (Figure 2).\n\n(A) Leymus mollis rhizome tissue. (B) Lawn grass (Festuca sp.) stem tissue (control). Ice seed crystals were placed in the extracts at slightly below the freezing point. Scale bars, 1 mm.\n\nScale bar, 1 mm.\n\nPrimers based on the IBPs of other pooid grasses succeeded in amplifying a sequence that encoded the C-terminal part of the IBP gene of L. mollis, including the entire IRI domain. The sequence (GenBank accession no. MT506011) encoded 260 a.a., which corresponds to all but the first 25 a.a. of the L. perenne IBP sequence. The sequence included the stop codon and contained no introns. Although the sequence was close to the sequence of L. perenne, it most closely matched an IBP-like protein from Triticum aestivum (QBE94480) (86% identity, 91% similarity), in agreement with L. mollis’s classification as a member of the Triticodae. When only the IRI domains were compared, the Leymus IRI domain was also closer to the Triticum IRI domain than to the Lolium IRI domain. The ice-binding activities of Tritium spp. IBPs have not yet been reported.\n\nThe IRI domain of L. mollis, like that of L. perenne, has eight repeats, each consisting of 14 or 15 residues (Figure 3A). The consensus sequences of the two IBPs are virtually the same (Figure 3A).\n\n(A) Comparison of the Leymus repeats (bottom) with those in the IRI domain of Lolium perenne (top). The Lolium data and the color scheme have been reproduced with permission from Middleton et al. (2012). Both domains have eight repeats. Consensus sequences are shown on top. Numbers indicate a.a. residue numbers. In the Lolium sequence, the gray background indicates the ice-binding site called the a side and the yellow background indicates a bulge on the b side. Similar features are found in the Leymus sequence. (B, C) Stereoviews predicted by SWISS MODEL using the IRI domain of L. perenne as template. SWISS MODEL was able to model the Leymus structure from Pro11 to Gly124, which corresponds to Asp1 to Ala118 in L. perenne. (B) View through the center of the coils, in which the ice-binding site (the a side) is on top. Amino acid side chains are shown only for the a and b sides. Color code of the ribbon: red, beta strand; cyan, coil. Color code of amino acid side chain: Cyan, C; blue, N, red, O; gray, H. (C) View of the b side of a space-filling model. A bulge is created by two residues on each of the first three coils. The first of the two residues in each coil is labeled. Colors are the same as those for the side chains in A.\n\nThe predicted structure of the L. mollis IBP IRI domain, obtained from using the L. perenne IBP IRI domain structure as template, resembles the Lolium IRI domain in several respects: it has eight repeats corresponding to eight coils in a beta-roll fold (Figure 3B); a flat side (the a side) that is populated with two rows that are rich in Thr and Ser residues (Figure 3B); an irregular side (the b side) that has a bulge in the first three coils (Figure 3C); and an interior that is dominated by Asn/His ladders (red residues in Figure 3A). The a side has been identified as the ice-binding site of L. perenne IBP. In Lolium, the average spacing between the alpha carbons of the row of amino acids on the ice-binding site is 4.5 Å, which is very similar to the spacing along the a-axis of ice (4.51 Å). In Leymus, the average spacing was calculated as 4.77 Å.\n\nIn summary, we describe an ice-binding protein from the grass Leymus mollis with ice-structuring and ice recrystallization inhibition (IRI) activities. The collection site of the grass, a gravelly beach on the Chukchi Sea, is an extreme habitat subject to numerous environmental stresses. L. mollis is also the highest latitude pooid grass so far examined for IBPs. Its IBP is similar in sequence to IBPs from other grasses and the predicted 3D structure of its IRI domain is very similar to that of the ryegrass Lolium perenne.\n\n\nData availability\n\nLeymus mollis 18S ribosomal sequence on GenBank, Accession number MT506010\n\nLeymus mollis IBP sequence on GenBank, Accession number MT506011\n\nFigshare: Ice structuring by ice-binding protein of Leymus mollis DSCN5945.JPG. https://doi.org/10.6084/m9.figshare.12401966.v1 (Raymond, 2020a)\n\nFigshare: Leymus mollis ice recrsytallization inhibition. https://doi.org/10.6084/m9.figshare.12401954.v1 (Raymond, 2020b)\n\nFigshare: PCR amplification of IBP and 18S genes of Leymus mollis. https://doi.org/10.6084/m9.figshare.12401957.v1 (Raymond, 2020c)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nJR thanks the School of Life Sciences, University of Nevada Las Vegas, for providing facilities for carrying out this study.\n\n\nReferences\n\nBalamurugan S, Ann JS, Varghese IP, et al.: Heterologous expression of Lolium perenne antifreeze protein confers chilling tolerance in tomato. J Integrative Agric. 2018; 17(5): 1128–1136. Publisher Full Text\n\nGagné JM, Houle G: Factors responsible for Honckenya peploides (Caryophyllaceae) and Leymus mollis (Poaceae) spatial segregation on subarctic coastal dunes. Am J Bot. 2002; 89(3): 479–485. PubMed Abstract \| Publisher Full Text\n\nJohn UP, Polotnianka RM, Sivakumaran KA, et al.: Ice recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic Antarctic hair grass Deschampsia antarctica E. Desv. Plant Cell Environ. 2009; 32(4): 336–348. PubMed Abstract\n\nKrieger E, Joo K, Lee J, et al.: Improving physical realism, stereochemistry, and side-chain accuracy in homology modeling: Four approaches that performed well in CASP8. Proteins. 2009; 77(Suppl 9): 114–122. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMiddleton AJ, Marshall CB, Faucher F, et al.: Antifreeze Protein from Freeze-Tolerant Grass Has a Beta-Roll Fold with an Irregularly Structured Ice-Binding Site. J Mol Biol. 2012; 416(5): 713–724. PubMed Abstract \| Publisher Full Text\n\nRaymond J: Ice structuring by ice-binding protein of Leymus mollis DSCN5945.JPG. figshare. Figure. 2020a. http://www.doi.org/10.6084/m9.figshare.12401966.v1\n\nRaymond J: Leymus mollis ice recrsytallization inhibition. figshare. Figure. 2020b. http://www.doi.org/10.6084/m9.figshare.12401954.v1\n\nRaymond J: PCR amplification of IBP and 18S genes of Leymus mollis. figshare. Figure. 2020c. http://www.doi.org/10.6084/m9.figshare.12401957.v1\n\nRaymond JA, Fritsen CH: Semipurification and ice recrystallization inhibition activity of ice-active substances associated with Antarctic photosynthetic organisms. Cryobiology. 2001; 43(1): 63–70. PubMed Abstract \| Publisher Full Text\n\nSandve SR, Rudi H, Asp T, et al.: Tracking the evolution of a cold stress associated gene family in cold tolerant grasses. BMC Evol Biol. 2008; 8: 245. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSidebottom C, Buckley S, Pudney P, et al.: Heat-stable antifreeze protein from grass. Nature. 2000; 406(6793): 256. PubMed Abstract \| Publisher Full Text\n\nWaterhouse A, Bertoni M, Bienert S, et al.: SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 2018; 46(W1): W296–W303. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "65703", "date": "20 Jul 2020", "name": "Peter L. Davies", "expertise": [ "Reviewer Expertise Protein biochemistry", "structural biology", "ice-binding proteins" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSformo and Raymond have prepared aqueous extracts of a hardy coastal grass (Leymus mollis) collected in Alaska and have observed the presence of an ice-binding protein (IBP) based on ice crystal faceting and the inhibition of ice recrystallization. From another extract they prepared DNA and used primers based on conserved regions in other grass IBP transcripts to amplify the L. mollis gene, which fortunately lacks introns. From the sequence of this DNA they were able to provide the amino acid sequence of the IBP domain involved in ice recrystallization. They used this to model the three-dimensional structure of the domain, which looks very much like that of Lollium perenne. Another set of primers was used to amplify a diagnostic section of the 18S ribosomal RNA gene whose sequence identified the species.\nThere is an element of comparative science here, and one would not need to see a repeat of this detailed analysis on other grasses/cereals. The presence of sequence homologs in transcriptomes as a way of seeing the distribution and variation of this IBP type should suffice from here on. Nevertheless, it is interesting to see this grass from a high latitude in Alaska (Beaufort Sea coast) extracted and analyzed. This report is also a good illustration of the use of molecular modeling to avoid solving a structure that would not differ much from the template. Indeed, the main features of the Lolium perenne IBP fold are retained in the L. mollis model, including the outward bulge of the beta-solenoid at one end on the non-ice-binding side.\n\nSuggestions for improvement:\nThe Abstract should include the percent identity of the IRI domain between the two grass species. This type of information should also be provided in the Introduction where the sequences of the IBPs of L. perenne and D. antarctica are compared. It is not helpful to say their sequences “are very similar”. In the last paragraph of the Introduction the L. mollis IBP sequence is again said to be “very similar” to those of the other two grasses. Please provide % identity and % similarity.\n\nFigure 1A shows ice structuring. However, since panel B also shows ice crystals it is important to point out to the average reader that the distinction lies in the faceting seen in A where flat, angular surfaces are the result of IBP binding to ice. The ice in B is smoother and rounded.\n\nFigure 2 is a rather poor presentation of ice recrystallization. One must look closely at the ice control images to see the grains are larger after 21 h. The methods say the ice recrystallization was done in water. From our experience the inclusion of 150 mM NaCl makes a huge difference to the visibility of the ice grains. The other suggestion is to take the starting picture at zero time with the ice held colder than – 3 °C. We see substantial recrystallization happening in the first hour, even at -6 °C, and will typically hold the ice wafer at -10 °C until the starting picture is captured, and then raise the temperature for more rapid recrystallization. I recommend that this analysis be repeated and improved.\n\nIn Methods, under ‘Structure prediction’, it is stated that three structures were proposed by SWISS MODEL operating with L. perenne IBP as a template. Then, the one with the ‘highest sequence identity’ was selected. Surely the sequence identity is the same for all models because they are based on the same template. We would typically compare models by root mean square deviation (RMSD) – how the atoms deviate from the model on average. Is this what is meant here?\n\nAlthough the sequence of the L. mollis open reading frame was incomplete, it would be useful to report the sequence upstream of the ice-binding domain. Presumably this is a series of leucine-rich repeats similar to those seen in other grass IBPs?\n\nIn reference to ice crystal axes the a- and c- should be italicized.\n\nReplace the contraction \"didn’t\" with \"did not\".\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5768", "date": "27 Jul 2020", "name": "James Raymond", "role": "Author Response", "response": "Percent identities added to Abstract. Identities were removed from Introduction to make a revision requested by reviewer 2.Fig. 1A: The sentence was revised to point out that the ice is smooth and rounded with no evidence of facet development.Fig. 2: The experiment requires much preparation and is difficult to repeat now that one of the authors is back in Alaska. However, we think there is a clear difference in recrystallization between the two samples when the figure is viewed at full scale.Swiss-Model: Swiss Model tries different regions of the the sequence to model, e.g., residues 131-247 in one model and residues 144-228 in another. Each gives a slightly different model. We selected the one that had the highest a.a. sequence identity.N-terminal region of IBP: For this, we would need a consensus sequence of the 5’ UTR in other grasses (for use as a forward primer) but we were unable to find such a sequence." }, { "c_id": "5778", "date": "17 Aug 2020", "name": "James Raymond", "role": "Author Response", "response": "Percent identity was added to Abstract and the Results. Identities were removed from Introduction to make a revision requested by reviewer 2.Fig. 1A: The sentence was revised to point out that the ice is smooth and rounded with no evidence of facet development.Fig. 2: We added a new control, Festuca (lawn grass) extract, which shows more clearly the greater RI activity of Leymus.Swiss-Model: Swiss Model tries different regions of the sequence to model, e.g., residues 131-247 in one model and residues 144-228 in another. Each gives a slightly different model. We selected the one that had the highest a.a. sequence identity.N-terminal region of IBP: For this, we would need a consensus sequence of the 5’ UTR in other grasses (for use as a forward primer) but we were unable to find such a sequence." } ] }, { "id": "65704", "date": "23 Jul 2020", "name": "Matthew Carlson", "expertise": [ "Reviewer Expertise Plant ecology and evolution - emphasis on Arctic and Boreal systems" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 brief report outlines a description of the sequence and structure of ice-binding protein in Leymus mollis from the Beaufort Coast in Arctic Alaska, and compares the findings to IBPs of other more temperate grass species.\nThe paper seems pretty straightforward, however, I do not have the expertise to comment on the amino acid sequence, protein modeling, and comparative approach to other species. The paper was well-written, the figures were informative, and I believe it warrants indexing.\nWhile this is a brief note, I do think that additional interpretation of results and providing some brief explanations related to mechanisms of freeze tolerance in light of their findings would make the manuscript substantially more valuable. I was not able to gauge if the similarities and differences between Leymus and Lolium/Festuca are likely to have physiological/ecological bearing on their respective environmental contexts. (As an aside, since Leymus grows all down the Pacific Coast to San Diego it would be interesting to know if the IBP structure or concentrations/ability to upregulate are different along that substantial environmental gradient.).\nI provide additional comments and edits embedded as notes in a PDF - please see the file here.\nI appreciate the opportunity to read this paper and I sincerely hope that my comments are useful in generating a more robust paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5767", "date": "27 Jul 2020", "name": "James Raymond", "role": "Author Response", "response": "Thank you for many good comments. Revisions have been made to address all of them." }, { "c_id": "5801", "date": "17 Aug 2020", "name": "James Raymond", "role": "Author Response", "response": "Thank you for many helpful comments. We revised the manuscript to address each of them. Our response to your comments can be viewed in the PDF file here." } ] }, { "id": "65705", "date": "27 Jul 2020", "name": "Hans Ramløv", "expertise": [ "Reviewer Expertise Cold tolerance of ectothermic animals", "antifreeze proteins", "cryptobiosis" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the present paper Sformo and Raymond present data on an ice binding and recrystallisation inhibiting protein from the Alaskan grass Leymus mollis, which lives in areas where it is exposed to several kinds of stresses including low temperatures.\n\nThe authors have observed ice structuring, recrystallisation inhibition activity in aqueous homogenates of the rhizome tissue. They have sequenced DNA from similar homogenates using primers based upon the conserved sequences from other IBP containing grasses. The obtained DNA sequences were translated into the amino acid sequences presumably involved in the ice recrystallisation inhibition and ice structuring. The amino acid sequence of the IRI domain of L. mollis was closely matched to the Triticum aestivum IRI domain.\n\nThe 3D structure of the ice binding domain was predicted using the obtained sequence and the structure of the IRI domain in Lolium perenne. It appears that the tree dimensional structure of the ice binding domain of L. mollis IBP quite resembles that of L. perenne.\n\nThe paper is an interesting comparative account of the ice related properties of a grass found at high latitudes - again indicating that ice structuring and recrystallisation inhibition is very important in plants adapted to cold climates.\n\nI miss an extension of the discussion of the comparative aspects of cold tolerance and adaptive aspects of the findings in the paper.\n\nFigure 1 could be explained a bit better as some readers may not understand the differences between the A and the B panel very well.\n\nFigure 2 is not very clear and also this figure would benefit from a bit more explanation in the text.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5789", "date": "17 Aug 2020", "name": "James Raymond", "role": "Author Response", "response": "• I miss an extension of the discussion of the comparative aspects of cold tolerance and adaptive aspects of the findings in the paper.We revised the last paragraph to give some perspective to the cold tolerance of L. mollis and how it probably differs from that of L. perenne.• Figure 1 could be explained a bit better as some readers may not understand the differences between the A and the B panel very well.We added some sentences to better explain the difference between Leymus and Festuca. • Figure 2 is not very clear and also this figure would benefit from a bit more explanation in the text.We added new photos to show RI of Festuca. It more clearly shows that Leymus has much stronger RI than Festuca." } ] } ]	1	https://f1000research.com/articles/9-648
https://f1000research.com/articles/9-992/v1	17 Aug 20	{ "type": "Research Article", "title": "SARS-CoV-2 meta-interactome suggests disease-specific, autoimmune pathophysiologies and therapeutic targets", "authors": [ "Gianmarco Bellucci", "Chiara Ballerini", "Rosella Mechelli", "Rachele Bigi", "Virginia Rinaldi", "Roberta Reniè", "Maria Chiara Buscarinu", "Sergio E. Baranzini", "Lohith Madireddy", "Giuseppe Matarese", "Marco Salvetti", "Giovanni Ristori", "Gianmarco Bellucci", "Chiara Ballerini", "Rosella Mechelli", "Rachele Bigi", "Virginia Rinaldi", "Roberta Reniè", "Maria Chiara Buscarinu", "Sergio E. Baranzini", "Lohith Madireddy", "Giuseppe Matarese", "Giovanni Ristori" ], "abstract": "Background: Severe coronavirus disease 2019 (COVID-19) is associated with multiple comorbidities and is characterized by an auto-aggressive inflammatory state leading to massive collateral damage. To identify preventive and therapeutic strategies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is important to ascertain the molecular interactions between virus and host, and how they translate into disease pathophysiology. Methods: We matched virus-human protein interactions of human coronaviruses and other respiratory viruses with lists of genes associated with autoimmune diseases and comorbidities associated to worse COVID-19 course. We then selected the genes included in the statistically significant intersection between SARS-CoV-2 network and disease associated gene sets, identifying a meta-interactome. We analyzed the meta-interactome genes expression in samples derived from lungs of infected humans, and their regulation by IFN-β. Finally, we performed a drug repurposing screening to target the network’s most critical nodes. Results: We found a significant enrichment of SARS-CoV-2 interactors in immunological pathways and a strong association with autoimmunity and three prognostically relevant conditions (type 2 diabetes, coronary artery diseases, asthma), that present more independent physiopathological subnetworks. We observed a reduced expression of meta-interactome genes in human lungs after SARS-CoV-2 infection, and a regulatory potential of type I interferons. We also underscored multiple repurposable drugs to tailor the therapeutic strategies. Conclusions: Our data underscored a plausible genetic background that may contribute to the distinct observed pathophysiologies of severe COVID-19. Also, these results may help identify the most promising therapeutic targets and treatments for this condition.", "keywords": [ "SARS-CoV-2", "COVID-19", "protein-protein interaction", "autoimmune disease", "interferon", "virus", "repurposing" ], "content": "Introduction\n\nThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current coronavirus disease 2019 (COVID-19) pandemic. The number of COVID-19 confirmed cases worldwide exceeds 14.5 million as of July 20, 2020, while the estimated global case fatality rate stands at 4.3%. Since the early phases of the pandemic, comorbidities such as hypertension, diabetes, cardiovascular disease, and obesity were readily identified as conditions associated with the severity of COVID-19 and the most aggressive collateral damage1,2. The acute respiratory distress syndrome was recognized as the condition that dominated the clinical picture in critically ill patients3.\n\nSARS-CoV-2 is the longest known ssRNA virus4. Like its predecessors SARS-CoV and MERS-CoV that caused epidemics in 2003 and 2012, SARS-CoV-2 is highly virulent. They all belong to the Coronaviridae family, together with four other HCoVs (HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1) that, in most cases, cause common colds. SARS-CoV-2 displays an overall 79% genome similarity with SARS-CoV, reaching a 99.7% homology within the Spike protein (S) gene, the major determinant of viral tropism5. In both viruses, the Spike protein binds the membrane protein angiotensin-converting-enzyme 2 (ACE2) and engages the serine protease TMPRSS2 to infect human cells6. The 0.3% genomic divergence in the critical (S)-Receptor Binding Domain7, and the potential to employ other proteases (e.g. FURIN)8 seem to explain the strikingly higher transmission capability of SARS-CoV-2.\n\nTo identify preventive and therapeutic strategies against COVID-19, it is important to ascertain the interactions between virus and host, and how they develop into disease pathophysiology. The definition of physical interactions between SARS-CoV-2 proteins and host proteins has advanced considerably in recent weeks9,10. In order to select relevant therapeutic targets and processes, it would be meaningful to pinpoint those interactions that are key to the development of the (most severe) clinical manifestations of the disease and understand their pharmacological relevance. Furthermore, if a plausible genetic association is identified, this significantly increases the probability of success in drug development11.\n\nA series of the clinical manifestations of COVID-19 appear to be related to an auto-aggressive immune dysregulation12. This evidence was clear enough to encourage the repurposing of drugs approved for the therapy of autoimmune conditions, for the treatment of severe COVID-19. It is therefore reasonable to hypothesize that the SARS-CoV-2-host interactome contains the genetic “seeds” and the therapeutic targets of the auto-aggressive processes that characterize COVID-19 (in particular the severe forms of the disease). To investigate both, we matched the SARS-CoV-2 interactome with lists of genes that genome-wide association studies (GWAS) defined as associated with major autoimmune diseases. We used this approach to also identify genes and processes that may influence the course of COVID-19 through the presence of coexisting diseases (i.e. matching the SARS-CoV-2 interactome genes with genes associated with COVID-19-relevant comorbidities).\n\nTo increase the interpretability and the specificity of our results, we also performed the above analyses using interactomes of the other coronaviruses (HCoVs) and other viruses causing respiratory diseases in humans.\n\n\nMethods\n\nWe considered the interactomes from 7 human infecting members of the Coronaviridae family and from other viruses causing respiratory diseases (Influenza A H1N1, Influenza A H7N9, Influenza B, HRSV) (Extended data13).\n\nThe SARS-CoV-2 host-protein network was recently uncovered by an affinity-purification mass spectrometry experiment9. Other host-pathogen protein interaction maps were derived from p-HIPSTER, a structure-informed atlas of human-virus interactions14, which contains both interactions present in the RCSB Protein Data Bank and predicted pathogen-host PPIs.\n\nWe performed the expansion using the latest upgrade of Human Integrated Protein-Protein Interaction rEference (HIPPIE)15 which integrates confidence scored and functionally annotated human protein-protein interactions (PPIs) from the public databases BioGRID, DIP, MIPS, HPRD, IntAct, MINT and BIND.\n\nTo guarantee the interaction confidence, we accounted only for physical interactions, starting from a very high PPI confidence score (0.95), and progressively expanded the network coverage until reaching a medium confidence score of 0.65 (second quartile of the HIPPIE score distribution).\n\nWe extracted, from the NHGRI-EBI Catalog of human genome-wide association studies16, the mapped genes of SNPs significantly associated (p-value ì≤ 5 × 10−8) with immune-mediated disorders [multiple sclerosis (MS), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Crohn’s disease (CD), ulcerative colitis (UC), type 1 diabetes mellitus (T1D)] and with conditions being relevant to COVID-19 clinical manifestations and prognosis [obesity, aging, type 2 diabetes mellitus (T2D), asthma, chronic obstructive pulmonary disease (COPD), hypertension and coronary artery disease (CAD)]. Specifically, we searched for the corresponding trait in the database and collected the reported loci (Extended data13).\n\nWe overlapped the interactomes and the disease-associated genes and evaluated the statistical significance using geneOverlap package version 1.24.017 in R, version 3.6.3. We took into consideration the Jaccard index, which represents the similarity between two sets; p-value significance cutoff was set at p<0.05 after correction with Benjamini-Hochberg method.\n\nWe selected the genes included in the statistically significant intersection of SARS-CoV-2 network and disease associated gene sets, constructing a COVID-19 meta-interactome. To better understand the immune mechanism shared by COVID-19 and immunological diseases, we also defined a COVID-19 immunological subnetwork collecting genes from immune-mediated disease only.\n\nWe then performed a functional enrichment analysis of each significant overlapping subset of genes among GO Biological Processes and Reactome Gene sets using Metascape18, a web-based utility. This tool uses the MCODE (Molecular Complex Detection) algorithm to perform module analysis and detect dense regions of protein interaction networks19. Network visualization and node connection analysis was performed with Cytoscape 3.8.020.\n\nThe Circle plot representing COVID-19 meta-interactome genes was produced using the Circos table viewer tool21. The UpSet plot was produced using the UpSetR package for R22, version 1.4.0.\n\nWe analyzed the COVID-19 meta-interactome genes using the GENE2FUNC of the FUMA tool23, an integrative web-platform to perform post-GWAS annotation. We specifically performed a gene expression analysis based on GTEx v8 and Ensemble version v92, retrieving tissue specificity. Gene-enrichment statistical analysis was set as follows: the adjusted P value cut-off was 0.05, minimum overlapping genes with gene-sets was 2, method for multiple test correction was Benjamini-Hochberg (FDR).\n\nWe looked for the expression of COVID-19 meta-interactome genes in samples derived from lungs of infected humans. To do this, we selected from the NCBI Gene Expression Omnibus Database24 the series GSE14750725, including transcriptional profiling of post-mortem lung samples of two male COVID-19 patients compared with healthy lung biopsies, and transcriptional profiling of normal human bronchial epithelial (NHBE) cells after the treatment with human IFN-I-Β.\n\nBriefly, at first we extracted the complete results using the GEO RNA-seq Experiments Interactive Navigator (GREIN) web tool26; we then selected the COVID-19 meta-interactome genes and plot the final data with EnhancedVolcano R package, version 1.4.0.\n\nTo assess the potential of COVID-19 meta-interactome genes of being regulated by Type 1 interferon-β, we exploited Interferome 2.0.1, an online database of interferon-regulated genes (IRGs)27. The search settings were as follows: Interferon type I, subtype IFN-β, any treatment concentration and time, in vitro and in vivo cell lines derived from Homo sapiens, any sample type, Fold change cutoffs 1.0.\n\nTo assess the therapeutic potential of COVID-19 meta interactome, we used the Scalable Precision Medicine Oriented Knowledge Engine (SPOKE)28. SPOKE is a heterogeneous knowledge network that includes data from 29 publicly available biomedical databases. We searched the SPOKE Neighborhood Explorer for the COVID-19 meta-interactome genes with the highest Betweenness Centrality score in the whole network and in subnetworks, filtering results with the following settings: “compound” and “gene” nodes; “compound-binds-protein”, “compound-downregulates-gene”, “compound-upregulates-gene”. We filtered nodes representing compounds at development phase ≥3; Edges Attributes and Limits were as set from the database.\n\n\nResults\n\nIn this work, we downloaded the host-protein networks of SARS-CoV-2, six other HCovS (SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1) and four other viruses causing respiratory diseases in humans (Influenza A H1N1, Influenza A H7N9, Inluenza B, Human Respiratory Syncitial Virus). We extended the experimentally determined SARS-CoV-2-host interactions9 by including second neighbors, i.e. molecules directly interacting with the host proteins engaged by the virus extracted from HIPPIE. We progressively lowered the confidence score threshold to construct 7 more sets of increasing size (Extended data13).\n\nWe then looked for an enrichment of human proteins interacting with SARS-CoV-2 within sets of genes mapped in genome-wide studies of immune-mediated conditions and comorbidities impacting on COVID-19 prognosis.\n\nThe analysis revealed a significant enrichement of SARS-CoV-2 interactome with genes associated with several autoimmune diseases (Figure 1). The strongest significance was reached by SARS-CoV-2/MS overlap (p=0.0002), consolidated at multiple expansion levels; significant associations with CD, SLE and RA were of weaker magnitude.\n\nWhen looking at clinically relevant comorbidities, there was a significant overlap between SARS-CoV-2 interactors and T2D associated genes, that followed a positive trend in parallel to the network expansion, with a maximum of 65 shared interactors (Figure 1 and Extended data13). The other COVID-19 relevant comorbidities reaching statistical significance were CAD and Asthma.\n\nThe heatmap shows the Jaccard Index of the overlaps of gene sets associated with immunological diseases and COVID-19 relevant comorbidities with viral interactomes. SARS-CoV-2 section shows in the first line the host protein network derived from experimental evidence9, followed by results obtained overlapping the expanded networks generated with HIPPIE settings at different confidence levels (0,95 to 0,65). HRSV: Human Respiratory Syncitial Virus; MS: Multiple Sclerosis; RA: Rheumatoid Arthritis; SLE: Systemic Lupus Erythematosus; CROHN: Crohn’s Disease. U.COLITIS: Ulcerative Colitis; CELIAC D.: Celiac Disease; T1D: Type 1 Diabetes Mellitus; T2D: Type 2 Diabetes Mellitus; CKD: Chronic Kidney Disease; COPD: Chronic Obstructive Pulmonary Disease; HTN: Hypertension; CAD: Coronary Artery Disease.\n\nNeither the other HCovs nor the other respiratory viruses (H1N1, H7N9, Influenza B, HRSV) reached significance levels as high as SARS-CoV-2.\n\nIn summary, SARS-CoV-2 host protein network is enriched in sets of genes associated with multiple autoimmune diseases and shares significant interactions with three conditions influencing COVID-19 prognosis: asthma, T2D and CAD.\n\nWe next constructed a COVID-19-human meta-interactome, selecting genes deriving from the significant overlaps with immunological diseases and comorbidities. The resultant network was composed of 248 genes (Figure 2A and Extended data13), with a non-specific distribution on autosomes, and a single gene located on Chromosome X (MECP2) and none in chromosome Y (Figure 2B).\n\nTo assess the prominence of interactors within the network, we ranked the gene according to the Betweenness score, a network centrality metric which reflects the number of shortest paths that go through a given node, thus representing the importance of that node on the information flow through the network (Figure 2C). Top ranking nodes were: MYC, a widely acting oncogene with a key role in immune regulation; XPO1, whose encoded protein mediates the nuclear export of cellular proteins and of RNAs, including viral ones; SMAD3, an intracellular transducer of transforming growth factor-β signaling pathway (Figure 2D). We then performed a functional enrichment analysis of each significant overlapping subset of genes using Metascape18 a web-based utility which uses the MCODE (Molecular Complex Detection) algorithm, to perform module analysis and detect dense regions of protein interaction networks19. Densely interconnected genes subsets pertain to central immunological processes (NFĸB, immune response and TLR3 pathway, podosome assembly, MAPK cascade), cell cycle and transcriptional regulation (TCF/LEF and WNT pathway), cholesterol and metabolism, and ionic channels regulation (Figure 3). In line with this data, the COVID-19 meta-interactome pathway enrichment analysis (Figure 4) showed that the top 20 clusters belonged to immunological (lymphocyte differentiation, neutrophils pathway, immune response-regulating signaling pathway, regulation of cytokine production), and cell cycle processes; interestingly, we found an enrichment of the hemostasis process. This is in line with evidence showing a coagulation dysfunction in severe COVID-1929\n\nA) Network Representation: node size is proportional to Betweenness Centrality score. B) Circle plot showing the chromosome distribution of the 248 COVID-19 meta-interactome genes. C) Top 10 nodes ranked by Betweenness Centrality D) Detail of the network view showing the top nodes.\n\nThe M-CODE algorithm was applied to detect densely interconnected node subsets. The network representation displays a different color for each cluster, associated with the enriched biologic processes in the relative boxes.\n\nHeatmap of top 20 enriched terms across input gene lists, colored by p-values. MS: Multiple Sclerosis; RA: Rheumatoid Arthritis; SLE: Systemic Lupus Erythematosus; CROHN: Crohn’s Disease. U. COLITIS: Ulcerative Colitis; CELIAC D.: Celiac Disease; T1D: Type 1 Diabetes Mellitus; T2D: Type 2 Diabetes Mellitus; CAD: Coronary Artery Disease.\n\nNotably, in COVID-19 meta-interactome composition, most of the genes deriving from autoimmune diseases’ sets appeared to be shared among the traits: the most relevant example is seen for MS and RA, whose SARS-CoV-2 interacting proteins derived from the same genes (specifically, 49 of the 61 GWAS-associated identical genes). These data suggested the presence of a “core” immune mechanism driving COVID-19 physiopathology shared with most of autoimmune conditions. Conversely, COVID-19 meta-interactome elements belonging to comorbidities’ subsets resulted more specific (T2D 52/66, CAD 12/20, asthma 29/61 unshared genes) (Figure 5A,B,C). This suggested the possibility that the pathobiological mechanisms underlying the worse prognosis in COVID-19 patients affected by these comorbidities are at least partly independent from the imbalanced immune response to SARS-CoV-2 infection.\n\nA) The table shows the subsets of genes from each trait contributing to the network composition. B) The circle plot shows the overlap between gene lists at the gene level, with purple curves linking identical genes. C) The UpsetPlot displays a schematic representation of the intersections among gene sets (x axis), with the respective size (y axis). MS: Multiple Sclerosis; RA: Rheumatoid Arthritis; SLE: Systemic Lupus Erythematosus; CROHN: Crohn’s Disease. U. COLITIS: Ulcerative Colitis; CELIAC D: Celiac Disease; T1D: Type 1 Diabetes Mellitus; T2D: Type 2 Diabetes Mellitus; CAD: Coronary Artery Disease.\n\nTo assess this hypothesis, we next performed a separate analysis of the immune subnetwork, containing genes derived from immunological diseases’ subsets (n=150), and the networks of each comorbidity (Asthma, n=61; T2D, n=66, CA D, n=20). The immune subnetwork pathway analysis confirmed a global enrichment of immunological functions. Betweenness Centrality ranking highlighted again MYC as the most relevant node, followed by RHOA, encoding for a small GTPase involved in cytoskeleton assembly and nuclear mechanotransduction, and TRAF6, a multiacting member of the toll-like receptor (TLR) family, capable of activating MAPK, PI3K, and interferon regulatory factor (IRF) pathways. These nodes also showed a high Betweenness Centrality score in the whole COVID-19 meta-interactome, thus highlighting the importance of immune processes in COVID-19 pathobiology. CAD and T2D only shared a few genes with the immune subnetwork, while, as expected, the overlap with asthma was more extended (Figure 6). Indeed, pathway and process enrichment analysis of asthma network showed the presence of immunological functions in the top clusters, in contrast to CAD (“extracellular structure organization”,”response to wounding”, “Plasma lipoprotein assembly, remodeling, and clearance”) and T2D (“Diseases of signal transduction by growth factor receptors and second messengers”, “Signaling by Receptor Tyrosine Kinases”, “PID HES HEY PATHWAY”) (Figure 7). In conclusion, comorbidities subnetworks are largely independent from the immune subnetwork, suggesting that distinct biological processes may interactively contribute to worsening COVID-19 clinical outcomes in these traits.\n\nT2D: Type 2 Diabetes; CAD: Coronary Artery Disease.\n\nEach square contains the network representation, the top nodes ranked by Betweenness Centrality (if >0,00) and the pathways with the highest enrichment. Node size is proportional to betweenness centrality; node color represents densely interconnected subsets.\n\nTo assess the tissue specificity of COVID-19 meta-interactome genes, we conducted a tissue expression pattern analysis based on GTEx v8 RNA-seq data exploiting the FUMA web tool. Results showed a significantly higher expression of COVID-19 meta-interactome genes in lungs, adipose tissue, blood vessels, blood, cervix and uterus. In contrast, the lowest expression was seen in liver, pancreas, kidney and heart (Figure 8A). Furthering the analysis at the gene level, we found a more prominent tissue specificity of subsets of genes: arteries showed a marked expression of ITGA8, ERG, IRS1; AGAP2, PKIA, GNG4, KCNJ3 resulted overexpressed in the brain, in contrast to the marked underexpression of MYC, AHR, CDK2, PLAU, TNFAIP3 in this tissue. Results also highlighted a core subset of genes with a marked expression throughout the whole body, as NFKBIA, STAT3 and FURIN, a protease known to contribute to SARS-CoV-2 S protein cleavage30 (Figure 8B). Overall, these expression patterns appeared to relate with the multifaceted clinical manifestation of the disease.\n\nA) Differentially Expressed Genes (DEGs) in GTEx v8 30 tissue types. Significantly enriched DEG sets (Pcorr < 0.05) are highlighted in red: the first graph shows DEGs overexpression, the second DEGs underespression, the third sums both side of regulation. B) Gene Expression Heatmap across GTEx v8 30 tissue types. Genes and tissues are ordered by clusters.\n\nWe then investigated if the expression pattern of COVID-19 meta-interactome genes was affected by SARS-CoV-2 infection in humans. To this aim, we reanalyzed the publicly available GSE147507 dataset, containing the transcriptional profiling of lung biopsies of a COVID-19 patient. SARS-CoV-2 infection caused an overall downregulation of COVID-19 meta-interactome genes, with the exception of a small group of genes including TNF (Figure 9A).\n\nRecent studies suggested that SARS-CoV-2 impairs type I Interferon production, hampering the host’s antiviral response while triggering a sustained chemokine storm10,25,31,32. To explore if the administration of IFN-β could revert the transcriptional profile of COVID-19 meta-interactome genes, we extracted from the GSE14750 the transcriptional profiling results of normal human bronchial epithelial cells treated with human recombinant IFN-β. Indeed, the COVID-19 meta-interactome genes showed a slight upregulation trend, in contrast to the effect of SARS-CoV-2, (Figure 9B). We then investigated the component of COVID-19 meta-interactome in the Interferome database, an online collection of experimental data regarding Interferon Regulated Genes (IRG): we retrieved experimental evidence that all the 248 COVID-19 meta-interactome genes are regulated by type I interferon.\n\nCOVID-19 meta-interactome gene expression in SARS-CoV-2 infected human lungs (A) and normal human bronchial epithelial cells treated with IFN-Β (B). Volcano plots displaying the gene expression patterns. The y axis shows adjusted p.value; the x axis shows the Fold Change.\n\nFinally, we investigated whether COVID-19 meta-interactome nodes were suitable targets for drug repositioning. With this aim, we interrogated the Scalable Precision Medicine Oriented Knowledge Engine (SPOKE)28, a continuously updated collection of biomedical data acquired from 25 databases, including CHEMBL, DrugBank and LINCS. We focused our search on the meta-hubs, i.e. the nodes with the highest Betweenness Centrality score in the whole meta-interactome and in the above analyzed subnetworks (Figure 2 and Figure 7), as their effects extend widely to the network, making them good candidates for therapeutic purposes. We only considered compounds in advanced stage of development (phase ≥3) and approved drugs, aiming at clinical actionability.\n\nAt first, we looked for drugs interacting directly with the protein derived from each gene and retrieved 10 proteins targeted by 66 different drugs (Extended data13) Among these appeared the JAK inhibitors Tofacitinib, approved to treat Rheumatoid Arthritis, and Fedratinib, developed for myeloproliferative diseases. This class of compounds is already under clinical investigation for COVID-19, owing to the capacity to interfere with SARS-CoV-2 infection of cells and to reduce cytokine hyperproduction33. Anti-TNF agents, included in our results, follow the same track by blocking a pivotal molecule of the hyperinflammatory response. Among less clinically explored compounds, is Niclosamide that proved to target both LYN and STAT3 in the meta-interactome: it is an approved anti-helminthic drug, which has been recently demonstrated to have very potent antiviral activity against SARS-CoV-234.\n\nConsidering the effects of SARS-CoV-2 infection on COVID-19 meta-interactome gene expression, we extended the screening in SPOKE to drugs capable of reverting the pattern for each top node. Among these we found anti-diabetic drugs (sitagliptin, pioglitazone, rosiglitazone, canaglifozin), statins (atorvastatin, fluvastatin, simvastatin) and an angiotensin-receptor-blocker (valsartan): these results enhance the pathological relation of COVID-19 with cardiovascular diseases and diabetes, with shared pathways that may benefit from the same interventions.\n\nResults also included antiviral agents, such as ribavirin, which is receiving positive results in COVID-19 management35, and nelfinavir, which has been demonstrated to inhibit SARS-CoV-2 infection and replication both in silico and in vitro36,37; along with these, anti-inflammatory, immunomodulatory and immunosuppressive agents also emerged. Of interest is the presence of progesterone, estradiol, selective estrogen receptor modulators (SERMs: Tamoxifen, Bazedoxifene, Raloxifene) and anti-androgen medications (finasteride, flutamide): these results suggest that the effects of diverse steroidal hormones at the transcriptional level may underlie the sex discrepancy seen in COVID-19 severity.\n\nIn conclusion, we found multiple repurposable molecules targeting SARS-CoV-2-host interactions: to organize the results, we implemented a “pleiotropy ranking” of the whole list of compounds, on the basis of the multiplicity of positive effects each drug is expected to have on COVID-19 meta-interactome both at the protein and at the gene expression level (Extended data13).\n\n\nDiscussion\n\nWe found a significant enrichment of SARS-CoV-2 interactors among proteins coded by genes associated with autoimmune diseases. Notably, similar enrichments were not found when we considered other HCoVs or other respiratory human viruses as controls. This finding suggests that SARS-CoV-2 is able to activate an auto-aggressive response in a subset of infected people and that such activation may be influenced by a genetic, virus-specific, predisposition of the host. The result has implications for risk-assessment and, possibly, for future vaccination policies.\n\nWe also found an enrichment of SARS-CoV-2 interactors among proteins coded by genes associated with comorbidities - T2D, asthma and CAD - that contribute to the severity of the disease. Interestingly, T2D and asthma have an inflammatory pathophysiology that is absent or less pronounced in those comorbidities that did not show clear enrichments. Also, in the case of T2D, asthma and CAD, the results seem to be specific for SARS-CoV-2 compared to other viruses.\n\nCompared to SARS-CoV-2, in the other highly pathogenetic H-CoVs the link with autoimmunity was definitely looser, pointing out to pathobiological differences between infections that share clinical similarities. The higher lethality rate of MERS-CoV (~30%) and SARS-CoV (~10%), the broader cytopathic effects across tissues, and the clearer tropism towards immune cells (macrophages, T lymphocytes, dendritic cells) configure them as ancestors struggling to adapt to the human host after the zoonotic transmission38–40. This is in line with the higher infectious rate of SARS-CoV-2, as well as with its divergent cytokine profile compared to other HCoVs41. Indeed, the exuberant immune response underlying the severe pneumonia and the multiorgan failure distinguishes COVID-19 from the other respiratory diseases caused by viruses42.\n\nWhen we considered the tissue expression of the COVID-19 meta-interactome in the GTEx resource, lung, blood and blood vessels emerged, in agreement with the main targets of the infection. Concerning the lung, the ‘in silico’ prediction found a significant correspondence with data ‘in vivo’ coming from a recent study on the transcriptome from infected patients25: most of the components of COVID-19 meta-interactome were down-regulated. This gene expression profile was consistent with an inappropriate inflammatory response (low levels of type I and III interferons, elevated chemokines and high expression of IL-6), suggesting reduced innate antiviral defenses coupled with exuberant inflammatory cytokine production. This pattern suggests that the expression of most components of COVID-19 meta-interactome in the lung is negatively affected by the infection and positively regulated by exogeneous type I interferon: this was confirmed by our reanalysis of transcriptional profiles of normal human bronchial epithelial cells treated with human recombinant interferon-β25, and by the presence of COVID-19 meta-interactors in the Interferome, the data set of interferon-regulated genes27. Moreover, the encouraging results of a recent trial with interferon β-1b in patients with COVID-19 support this view35.\n\nIt is intriguing to note that interferon β is a pivotal first-line therapy for MS43, the autoimmune disease with the strongest enrichment in SARS-CoV-2 interactors. It will be interesting to verify the clinical course of MS patients infected by SARS-CoV-2, receiving IFN-β or other immunotherapies44.\n\nThe construction of a COVID-19 meta-interactome allowed us to identify densely interconnected clusters of interactors encompassing gene products of autoimmune diseases and comorbidities, and highly connected nodes, exerting a critical control on the network. These meta-hubs, mainly linked to immunological pathways (immune response regulation, cytokine production, interleukin signaling, neutrophils pathway) may represent clinically actionable interactors, suitable for anti-virus drug discovery or repurposing. Among the main pathways resulting from the analysis of the meta-interactome, we found an enrichment for hemostasis pathways. This result supports the dysfunction of coagulation that is frequently observed in complicated cases of SARS-CoV-2 infection. Our recent analysis on genome-wide MS association data and coagulation showed an over-connectivity between the two networks45, suggesting that the link between hyper-inflammatory state and activation of coagulative pathway is shared by COVID-19 and MS.\n\nThe presence of more independent sub-networks of T2D and CAD within the meta-interactome suggests the existence of two mechanisms underlying COVID-19 course: one is autoinflammatory, SARS-CoV-2-specific, and under the influence of the genetic background of the host; the other is influenced by the presence of other diseases that synergize with the virus in a non-specific way as they do with many other microbial and non-microbial diseases.\n\nThe drug repurposing screening we performed to target COVID-19 meta-interactome key nodes suggest the possibility to tailor the therapeutic framework including drugs targeting the immune subnetwork together with compounds interfering with the specific comorbidity subnetwork.\n\nOur analysis presents some limitations. First, the lack of a SARS-CoV-2 interactome integrating predicted and confirmed protein-protein interactions (PPIs) – as for the other viruses we considered – caused us to use a different method to expand the in vitro demonstrated network. To homogenize this mismatch, we included in the analysis multiple levels of high-confidence, only physical PPIs inferred by an optimally referenced bioinformatic resource. Additionally, we built our analysis at the gene level, and the single-nucleotide variants specifically associated with each of the considered conditions may affect the resultant virus-host interaction, either in a positive or a detrimental fashion.\n\nGenome-wide association studies, as well as genetic profiling of rare naturally-resistant individuals and “unexpectedly” severe cases, are needed to clarify the possible role played by the host genetic variability in the clinical outcome of SARS-CoV-2 infection46,47. In this context, recent results highlighted 2 loci associated to respiratory failure in COVID-1948, that were not included in our analysis. However, the proteins coded by genes associated to these loci, showed direct interaction with some members of our meta-network: FYCO1 with XPO1, EP300, CYLD; LZTFL1 with CEP25049–52. Indeed, our study complements these approaches, proxying genetics to clarify COVID-19 pathophysiology, and provides information that may increase the probability of success in the development of new treatments.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nOpen Science Framework: SARS-CoV-2 meta-interactome suggests disease-specific, autoimmune pathophysiologies and therapeutic targets: extended data. https://doi.org/10.17605/OSF.IO/82T5Q13\n\n- SARS-COV-2 meta-interactome genes list.csv (List of interacting genes)\n\n- SARS-COV-2 meta interactome extended data.xlsx (Viral interactomes size and sources, gene sets compositions, gene sets/interactomes intersections numbers, meta-interactome composition, drug target screening and drug pleiotropy ranking)\n\n- Viral interactomes composition..csv (Composition of viral interactomes)\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\nZhou F, Yu T, Du R, et al.: Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLa Starza S, Ferraldeschi M, Buscarinu MC, et al.: Genome-wide multiple sclerosis association data and coagulation. Front Neurol. 2019; 10: 95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCasanova J, Su HC, Human C, et al.: A global effort to define the human genetics of protective immunity to SARS-CoV-2 infection. Cell. 2020; 181(6): 1194–1199. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGanna A, Unit TG, General M: The COVID-19 Host Genetics Initiative, a global initiative to elucidate the role of host genetic factors in susceptibility and severity of the SARS-CoV-2 virus pandemic. Eur J Hum Genet. 2020; 28(6): 715–718. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEllinghaus D, Degenhardt F, Bujanda L: Genomewide Association Study of Severe Covid-19 with Respiratory Failure. N Engl J Med. 2020; NEJMoa2020283. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "69604", "date": "14 Sep 2020", "name": "Siddhartha Mahanty", "expertise": [ "Reviewer Expertise Parasite immunology", "Immuneregulation", "viral hemorrhagic fevers", "antibody functions" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have conducted a comprehensive interactome analysis of SARS-CoV-2 infections in humans using a number of conventional and recently developed computational approaches. Genes and pathways of significance identified by this interactome analysis were matched with genes associated with comorbid disease (type-2 diabetes, cardiovascular disease, chronic obstructive pulmonary disease etc) using a similar approach to identify a meta-interactome between COVID-19 disease and the comorbidities associated with disease severity. They demonstrate that the SARS-CoV-2-host interactors concentrated in immunological pathways and were strongly associated with autoimmunity and three comorbidities of prognostic significance, diabetes, coronary artery disease, and asthma. Analysis of RNAseq data from post-mortem lung tissue in a single patient demonstrated lower expression of meta-interactome genes identified in their network analysis. Finally, the authors speculate on potential drug targets, among currently used drugs in the autoimmunity and diabetes domains that may be of interest for the management of SARS-CoV-2 infections.\n\nThe rationale for the computational approach, selection of analysis software, and methodology used in the network analysis are described adequately, although I would recommend clearer explanations with some background information about each approach for the general readership unfamiliar with this field. The results are consistent with the proposed pathophysiology of COVID-19 disease, and there are no real surprises in this exploratory research. This raises a question as to the importance of the sample size from which they have available data. How much could a larger sample size, particularly samples from infected individuals with a broader range of clinical presentations influence their results? Many conclusions appear to be speculative or based on small (in some cases, single) studies and patient numbers – bias is frequently unavoidable in such small, select datasets. These will presumably require validation with larger data sets. Nevertheless, the comprehensive meta-interactome undoubtedly serves as a good framework for a generation of hypotheses and starting points for research on pathogenesis and treatment of COVID-19 disease.\n\nGeneral comments on presentation and content:\nMuch of the data presented in figures are direct outputs of computational analysis. The manuscript would benefit from more creative displays for the general readership, such as a schematic encompassing the study design and methodology and the type of data generated with each procedure.\n\nIn describing the analysis of interactomes between SARS-CoV-2 and disease-associated genes (end of Page 3) the term “overlapped” is unclear. Do they mean genes that are regulated similarly in the two conditions? A clearer explanation of the procedure is needed for readers unfamiliar with interactome methodology.\n\nWhile mention is made of the 6 other coronaviruses and 4 other respiratory viruses as reference organisms, how they were used in the analysis is unclear.\n\nSuggestions to the research community about the type of data that are required to fill knowledge gaps or refine the interactome would be helpful in directing ongoing research efforts.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "72616", "date": "19 Oct 2020", "name": "Mohd M. Khan", "expertise": [ "Reviewer Expertise Systems Proteomics", "Host-pathogen Biology of Infectious Diseases" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study, the authors conducted (meta) interactome analyses to deduce the molecular interactions between the host and virus that shape the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans. Such analyses were further leveraged to uncover the molecular pathways and associated genes that may play roles in co-morbidities (e.g., type 2 diabetes (T2D), coronary artery diseases (CAD), immune-related conditions, and respiratory diseases such as asthma) that may impact disease severity and overall outcomes. The authors concluded that a significant enrichment of SARS-CoV-2 interactome was tied to the genes that have established roles in multiple autoimmune diseases, and share significant interactions with CAD, T2D, and asthma co-morbidities. Furthermore, they conducted in silico drug target screening and proposed several molecules that may target SARS-CoV-2-host interactions and inflammatory pathways associated with infection. To validate some of the meta-interactome genes identified in their analysis, authors leveraged the RNAseq data ((GEO: GSE147507; data is from a peer-reviewed study that proposed that the reduced innate antiviral defenses (low levels of type I and III interferons (IFNs)) and inflammatory cytokine production (elevated chemokines and high expression of IL-6) may drive COVID-19 infection) and found reduced expression of meta-interactome genes in human lungs post-SARS-Cov2-infection and proposed roles of Type I IFNs.\nOverall, this exploratory study is interesting as reported results are in conformity with the earlier published work on coronaviruses and their associated pathophysiologies; introduction, methodology, results, and discussion sections are well-described and appropriately cited. Given there is additional information available, including on the potential of IFN therapies (stand-alone and/or in a combination with other drugs; eg., ACTT-3 trial), authors may cite those and provide additional context for the exploratory results provided here.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "76689", "date": "07 Jan 2021", "name": "Yongming Sang", "expertise": [ "Reviewer Expertise Immunogenetics", "antiviral immunology", "interferon biology", "immunometabolism" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript describes a computational study of SARS-CoV2 interactome and its association with comorbid situations, especially several autoimmune diseases. The findings from different datasets, converge a causal relationship of SARS-CoV2 infection, particularly during severe COVID-19 progression, with an autoimmune regulation as shown in several autoimmune diseases. Although this kind of autoimmune situation has been associated with COVID-19 progression in previous studies, a meta-interactome study as presented strengths the observation and is informative for targeting prophylactic design and drug repurposing as indicated. I pretty much agree with the second reviewer regarding the pitfalls of presentation and improving readability. Additional concerns include:\nThe interactome varies during the different stages of COVID-19 progression, the autoimmune manifestation is mostly associated with severe COVID-19 patients. The enrichment of interactome loci as shown in Figure 1 is need to correlate to the status of COVID-19 severity (Such as classified in a Yale IMPACT study Lucas C., et al. Nature 584, 463–469 (2020)).\n\nThe description of SARS-CoV2’s suppression of interferon response does not well reflect the full scenario of dysregulated interferon response during the viral infection and COVID-19 progress. Several studies actually determined robust interferon response COVID-19 patients. This also reflected by all the 248 meta-interactome genes are IRGs. Please refer to some further references to revise the relevant description:\nLopez L, Sang PC, Tian Y, Sang Y. Dysregulated Interferon Response Underlying Severe COVID-19. Viruses. 2020 Dec 13;12(12):1433.1\nLee, J.S.; Park, S.; Jeong, H.W.; Ahn, J.Y.; Choi, S.J.; Lee, H.; Choi, B.; Nam, S.K.; Sa, M.; Kwon, J.S.; et al. Immunophenotyping of COVID-19 and influenza highlights the role of type I interferons in development of severe COVID-19. Sci. Immunol. 2020, 5, eabd1554.2\nLucas, C.; Wong, P.; Klein, J.; Castro, T.B.R.; Silva, J.; Sundaram, M.; Ellingson, M.K.; Mao, T.; Oh, J.E.; Israelow, B.; et al. Longitudinal analyses reveal immunological misfiring in severe COVID-19. Nature 2020.3\n\nOther recent GWAS studies have not been included for analysis and discussion. In addition to gene loci in IFN-signaling, multiple gene loci in chemokine signaling were reproducibly associated with COVID-19. The following key references need to be considered for interpreting/discussing their in silico findings:\nMcCoy, K.; Peterson, A.; Tian, Y.; Sang, Y. Immunogenetic Association Underlying Severe COVID-19. Vaccines 2020, 8, 700.4\nSevere Covid-19 GWAS Group; Ellinghaus, D.; Degenhardt, F.; Bujanda, L.; Buti, M.; Albillos, A.; Invernizzi, P.; Fernández, J.; Prati, D.; Baselli, G.; et al. Genomewide Association Study of Severe Covid-19 with Respiratory Failure. N. Engl. J. Med. 2020, 383, 1522–1534.5\nPairo-Castineira, E.; Clohisey, S.; Klaric, L.; Bretherick, A.; Rawlik, K.; Parkinson, N.; Pasko, D.; Walker, S.; Richmond, A.; Fourman, M.H.; et al. Genetic mechanisms of critical illness in Covid-19. medRxiv 2020.6\nZeberg, H.; Pääbo, S. The major genetic risk factor for severe COVID-19 is inherited from Neanderthals. Nature 2020.7\n\nMinor issues:\n\n‘drug repurposing’ is a more meaningful keyword; “impairs (or low levels of) type I Interferon production” are not accurate. Please see Point 2 above. Please define and make consistent the abbreviations used in the text. Such as “H-CoV/HCoV”.\n\nFigure 8A, what is the legend for either blue bars or red bars. Seems the same thing, and does DEG for both sides mean total DEG?\n\nAs indicated all 248 interactome gene loci are IRGs, i.e. IFN signaling is the core of the profiled interactome, why the drug repurposing study identifies no IFN-based prophylactics?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-992
https://f1000research.com/articles/9-238/v1	03 Apr 20	{ "type": "Research Article", "title": "Burden of disease due to hip, knee, and unspecified osteoarthritis in the Peruvian social health insurance system (EsSalud), 2016", "authors": [ "Roger V. Araujo-Castillo", "Carlos Culquichicón", "Risof Solis Condor", "Carlos Culquichicón", "Risof Solis Condor" ], "abstract": "Introduction: Since its introduction by the World Health Organization (WHO), the concept of burden of disease has been evolving. The current method uses life expectancy projected to 2050 and does not consider age-weighting and time-discounting. Our aim is to estimate the burden of disease due to hip, knee, and unspecified osteoarthritis using this new method in the Peruvian Social Health Insurance System (EsSalud) during 2016. Methods: We followed the original 1994 WHO study and the current 2015 Global Burden of Disease (GBD) methods to estimate disability adjusted life years (DALY) due to osteoarthritis, categorized by sex, age, osteoarthritis type, and geographical area. We used disability weights employed by the Peruvian Ministry of Health, and the last update issued by WHO. Results: Overall, EsSalud reported 17.9 new cases of osteoarthritis per 1000 patients per year. Annual incidence was 23.7/1000 among women, and 72.6/1000 in people above 60 years old. Incidence was 5.6/1000 for knee osteoarthritis and 1.1/1000 for hip. According to the 1994 WHO method, there were 399,884 DALYs or 36.6 DALYs/1000 patients per year due to osteoarthritis. 12.4 and 2.2 DALYs/1000 patients per-year were estimated for knee and hip osteoarthritis, respectively. Using the 2015 GBD method, there were 1,037,865 DALYs or 94.9 DALYs/1000 patients per year. 31.4 and 5.3 DALYs/1000 patients per year were calculated for knee and hip osteoarthritis, respectively. Conclusions: In the Peruvian social health insurance subsystem, hip, knee, and unspecified osteoarthritis produced a high burden of disease, especially among women and patients over 60. The 2015 GBD methodology yields values almost three times higher than the original recommendations.", "keywords": [ "Osteoarthritis", "burden of disease", "disability adjusted life years", "Peru", "social inssurance" ], "content": "Introduction\n\nGlobally, the musculoskeletal disorders are a group of chronic diseases with high disability rates, which have been increasing, especially among people above 60 years old1,2. Worldwide, osteoarthritis is the sixth cause of disability-adjusted life years (DALYs), representing 3% of global burden of disease3. Knees and hip osteoarthritis produce the greatest disability in patients, with 6%, and 4% of the burden of disease due to knee and hip osteoarthritis, respectively1,2. Overall, the prevalence of osteoarthritis is homogeneous among men and women between 30 to 60 years old, but increases among women after that4,5.\n\nThe Peruvian burden of disease studies estimated that 308,804 DALYs were lost in the Peruvian population due to musculoskeletal disorders, representing 6% of the disease burden from all health conditions during 20046,7. Osteoarthritis was the health condition with the seventh greatest disability rates and caused 165,636 DALYs, which represents 3% of the total disease burden6,7. 98% of DALYs from osteoarthritis were attributed to the years lost due to disability (YLD), since osteoarthritis is not a primary cause of death. It mostly affected women, being the fourth biggest cause of DALYs in women6. Among people between 45 and 59 years old, osteoarthritis was the second biggest cause of disability, with 109,804 DALYs, and in people above 60 years old represented 7% of total DALYs7–9. Without knowing the impact of osteoarthritis on people's lives, it is difficult to propose solutions, invest resources for prevention, and mitigate disability, especially among elders, who constitute a large group within the Peruvian social health insurance system (EsSalud), which cares for approximately 37% of people who seek medical attention10,11. Hence, there is an important need to estimate the burden of disease produced by osteoarthritis in this healthcare subsystem11.\n\nSince its introduction by the World Health Organization (WHO), the concept of burden of disease has been evolving. The original 1994 recommendations used tables based on 1966 data, employed different life expectancy for men and women, gave less weight to the extremes of life, and penalized years when they were far from the current person age12. The current method recommended by WHO in its 2015 Global Burden of Disease (GBD) study uses life expectancy projected to 2050 and does not consider age-weighting or time-discounting13. Depending on the age distribution of a population, these new recommendations would probably yield higher estimates. Therefore, our aim is to estimate the burden of disease due to hip, knee, and unspecified osteoarthritis in EsSalud during 2016 using two different methods.\n\n\nMethods\n\nOur study was conducted using nationwide data collected by the Peruvian social health insurance system during 2016. We included records of all patients older than 15 years who were attended due to osteoarthritis (International Classification of Diseases, ICD-10: M15-M19) between January and December 2016. We excluded patients whose ICD-10 codes for osteoarthritis were registered during hospitalization and patients who were previously treated for osteoarthritis. Sample size calculation estimated that a minimum of 20,000 records were needed to find differences of at least four DALYs/1000 people between subgroups with 80% power and 95% significance.\n\nAll medical attentions at EsSalud between January and December 2016 were reviewed. These attentions had been registered using three different electronic systems. The Hospital Management System (SGH, by its acronym in Spanish) and the Health Services Management System (HSS, by its acronym in Spanish) record inpatient and outpatient attentions delivered at secondary and tertiary care level facilities. Meanwhile, the Health Information System for Primary Care Centers (SISCAP, by its acronym in Spanish) registers health attentions delivered at primary care facilities. All entries that fulfilled our selection criteria were included. In addition, we reviewed all death certificates issued by EsSalud during the study period, and selected those patients who had osteoarthritis registered as primary or contributory cause of death. Identities of patients on databases were kept confidential during data management and analysis.\n\nCovariates included sex, age, health center, geographical area, osteoarthritis of the hip (ICD-10: M16), osteoarthritis of the knee (ICD-10: M17), osteoarthritis of first carpometacarpal joint (ICD-10: M18), polyosteoarthritis (ICD-10: M15), unspecified osteoarthritis (ICD-10: M19), death due to osteoarthritis, and time between initial attention for osteoarthritis and death. Additionally, the following indicators were estimated for each person:\n\nLife expectancy at time of care delivered: Life expectancy was estimated according to two methods: The original WHO burden of disease study from 19945 employed the West extended model life tables from level 2612 to calculate life expectancy at the time the disability started (in our case, time of osteoarthritis diagnosis). It then applied an age-weighted function, defined by: Cxe-βx, where “x” is the age in years, “C” is the constant of age weighting adjustment (value: 0.16458) and “β” is the age weighting parameter (value: 0.04). This function draws a curve assigning different weights to ages, giving greater values to adult ages since they were considered “more productive”. Additionally, a 3% discount by year was applied, trying to capture the fact that people appreciate years in the immediate future than those further away more. The second method, employed in the 2015 GBD study, uses the maximum worldwide life expectancy projected to 2050, does not differentiate between men and women, and does not consider weights or discounts13.\n\nYears of life lost (YLL): In case the patient died because of osteoarthritis, the YLL was calculated as the life expectancy at the time of death using the two methods described above12,13. Living patients were assigned a zero value for YLL.\n\nYears lost due to disability (YLD): YLDs were estimated as the average duration of the illness at age of onset, times the disability weight (0 = maximum health, 1 = death)12. Given that osteoarthritis is a chronic condition that lasts until death, the average duration of the illness was considered as the life expectancy at the time of initial diagnosis. For this estimation, we used the two methods previously described12,13. Two disability weights were used: 0.165, which is the value employed by the 2015 GBD study for severe musculoskeletal diseases of lower limbs14,15; and 0.28, which was used in the Peruvian burden of disease studies for osteoarthritis severe enough to seek medical attention6–10.\n\nDisease-adjusted life years (DALY): This was the sum of the years of life lost (YYL) and the years lost due to disability (YLD) for each patient.\n\nWe described numerical variables using means and standard deviations. Categorical variables were described using frequencies and proportions. Osteoarthritis incidences were calculated by dividing the number of new cases registered by the number of insured patients in EsSalud during 2016. YLL, YLD, and DALYs were estimated, summing these metrics in total and by subgroups using the 1994 WHO and the 2015 GDB methods, and the 2015 GBD and the Peruvian Ministry of Health (MINSA) disability coefficients, which means four iterations were calculated for each metric. In addition, DALYs incidence ratios per thousand patients per year were calculated by dividing total number of DALYs by the total number of insured patients registered in EsSalud during 2016. The statistical analysis used STATA v14.0 (Statacorp, College Station, Tx). Code used for the analysis is available as Extended data16.\n\nThe Institutional Review Board (IRB) of the Edgardo Rebagliati Martins National Hospital (HNERM) approved this study (#832-2019-195). The IRB waived the requirement for consent from the patients as the study was conducted with an anonymized dataset.\n\n\nResults\n\nDuring 2016, the Peruvian social health insurance system attended 196,003 patients for a first time visit due to osteoarthritis. Among them, 65.5% (n=128,323) were women, the mean age was 60.9±15.1 years, 62.6% (n=122,705) had polyosteoarthritis or unspecified osteoarthritis, 31.0% (n=60,788) had osteoarthritis of the knee, 5.9% (n=11,472) osteoarthritis of the hip and 0.5% osteoarthritis of other joints17. We estimated 17.9 new cases of osteoarthritis per 1000 insured patients in 2016. The incidence of osteoarthritis in women was 23.7/1000 insured patients per year, and in patients over 60 years old the incidence was 72.6/1000 insured patients per year. The incidence of polyosteoarthritis or unspecified osteoarthritis was 11.2/1000 patients per year, the incidence of knee osteoarthritis was 5.6/1000 patients per year, and hip 1.1/1000 patients per year. The geographical region with higher incidence was the Northern Coast/Highlands with 20.2/1000 patients per year, and the lowest was found in the Amazon Rainforest with 13.3/1000 patients per year (Table 1).\n\nSince only three deaths certificates registered osteoarthritis as the primary cause of death, the DALYs corresponded mainly to the years lost due to disability (YLD). According to the 1994 WHO methodology, osteoarthritis produced 399,884 DALYs using the latest WHO disability weights, or 678,591 DALYs if employing the Peruvian MINSA weights. Using these disability weights and after adjusting by the number of insured patients, osteoarthritis delivered 62.0 DALYs/1000 patients per year, poly/unspecified osteoarthritis 37.0 DALYs/1000 patients per year, knee osteoarthritis 21.0 DALYs/1000 patients per year, and hip osteoarthritis 3.8 DALYs/1000 patients per year (Table 2). According to the 2015 GBD methodology, osteoarthritis produced 1,037,865 DALYs using the latest WHO disability weights, or 1,761,225 DALYs if employing the Peruvian MINSA weights. Using these disability weights and after adjusting by the number of insured patients, osteoarthritis delivered 161.0 DALYs/1000 patients per year, poly/unspecified osteoarthritis 97.2 DALYs/1000 patients per year, knee osteoarthritis 53.3 DALYs/1000 patients per year, and hip osteoarthritis 9.7 DALYs/1000 patients per year (Table 2).\n\nDALY, disability-adjusted life year; WHO, World Health Organization; GBD, Global Burden of Disease; MINSA, Peruvian Ministry of Health\n\nWe analyzed the burden of disease by knee and hip osteoarthritis subgroups. For knee osteoarthritis, the incidence was 4.5 new cases/1000 patients per year among men and 6.7 new cases/1000 patients per year in women. People between 15–44 years old had only 2.4 new cases/1000 patients per year, whereas people aged 60 or over presented 20.2 new cases/1000 patients per year. According to the 1994 WHO methodology and using the Peruvian MINSA disability weights, knee osteoarthritis produced 16.6 DALYs/1000 patient per year among men, and 25.5 DALYs/1000 patient per year in women. It was also responsible for 17.1 DALYs/1000 patient per year among people 15–44 years old, and 38.9 DALYs/1000 patient per year in patients over 60 years old (Table 3).\n\nDALY, disability-adjusted life year; WHO, World Health Organization; GBD, Global Burden of Disease; MINSA, Peruvian Ministry of Health.\n\nRegarding hip osteoarthritis, the incidence was 0.7 new cases/1000 patients per year among men and 1.4 new cases/1000 patients per year in women. People between 15–44 years old had only 0.4 new cases/1000 patients per year, whereas people aged 60 or more presented 4.1 new cases/1000 patients per year. According to the 1994 WHO methodology and using the Peruvian MINSA disability weights, knee osteoarthritis produced 2.3 DALYs/1000 patients per year among men, and 5.3 DALYs/1000 patients per year in women. It was also responsible for 3.0 DALYs/1000 patients per year among people 15–44 years old, and 7.6 DALYs/1000 patient per year in patients over 60 years old (Table 3).\n\n\nDiscussion\n\nThe incidence for osteoarthritis overall was 17.9 new cases/1000 insured patients per year, for poly/unspecified osteoarthritis was 11.2/1000 patients per year, for knee osteoarthritis was 5.6/1000 patients per year, and for hip osteoarthritis was 1.0/1000 patients per year. These findings are consistent with estimations previously made in Peru and high-income countries. In Peru during 2009, the incidence of knee osteoarthritis was 3.26/1000 inhabitants per year, and for hip osteoarthritis was 0.91/1000 inhabitants per year8. In Canada, the incidence of all osteoarthritis was 8.6 new cases/1000 inhabitants per year with a prevalence of 80.3 cases/1000 inhabitants until 201518. In Spain there was an incidence of 6.5 new cases/1000 inhabitants per year for knee osteoarthritis and 2.1/1000 inhabitants per year for hip osteoarthritis during 201419. The findings of the 2010 GBD Study estimated a prevalence of 38 cases/1000 inhabitants per year for knee osteoarthritis and 8.5 cases/1000 inhabitants per year for hip osteoarthritis20.\n\nConsidering MINSA had used the 1994 WHO methodology and their own disability weights in their previous burden of disease studies, we will follow suit in order to compare our results with previous reports. Hence, the estimated burden for overall osteoarthritis was 62.0 DALYs/1000 patients per year, for poly/unspecified osteoarthritis was 37 DALYs/1000 patients per year, for knee osteoarthritis was 21 DALYs/1000 patients per year, and for hip osteoarthritis was 3.8 DALYs/1000 insured patients per year21. These estimates are consistently higher than the disease burden of osteoarthritis reported by all studies conducted in Peru under the 1994 WHO methodology. Since 2006, the General Directorate of Epidemiology (DGE, by its acronym in Spanish) of MINSA, which has carried out studies to estimate the osteoarthritis disease burden in Peruvian settings, initially estimated 94,160 DALYs due to osteoarthritis (3.4 DALYs/1000 inhabitants)3. For 2009, the DGE estimated 165,636 DALYs due to osteoarthritis7. In 2012, osteoarthritis delivered 193,774 DALYs (6.4 DALYs/1000 inhabitants)9. In 2015, EsSalud carried out a study of the burden of disease among the insured patients and estimated 131,220 DALYs due to osteoarthritis (12.3 DALYs/1000 insured)10.\n\nAs observed in Table 4, there has been an upward trend in the DALY estimates despite using the same methodology. This could be due to different approaches to measure incidence. Previous studies have used a variety of sources including epidemiological surveillance, number of attentions, population-based surveys, medical chart reviewing, and scientific papers6–10. It is possible then, that the upward trend is just echoing a better registry of cases. In the present study, we only used electronic records of attentions, and employed all national data instead of small samples. This could lead to better capturing of cases, otherwise overlooked in previous studies. On the other hand, it is possible that our data collection strategy overestimates the number of new cases. Since we based our estimations on the ICD-10 diagnosis entered for each attention, it is not certain that all cases were confirmed at the time of the medical visit. In addition, we could have included mild cases not usually considered for surveillance or research purposes.\n\nDALY, disability-adjusted life year; WHO, World Health Organization; MINSA, Peruvian Ministry of Health; EsSalud: Peruvian social health insurance system; ICD, International Classification of Diseases.\n\nWe also observed discrepancies when stratified by age group. In our study, the estimated DALYs were higher for insured patients between 45–59 years old, with 150.0 DALYs/1000 patients per year, followed by people older than 59 years, with 134.8 DALYs/1000 patients per year, and people between 15–44 years old, with 42.8 DALYs/1000 patients per year. In contrast, the 2012 Peruvian study found that the elderly group produced the most DALYs/1000 people9. The same study found that the elderly group delivered a burden of disease almost nine times the one registered for 15–44 years old people (26.0 vs 3.0 DALYS/1000 inhabitants); meanwhile, our study found a much more reduced gap of only three times (134.8 vs 42.8 DALYS/1000 inhabitants). One possibility is that our study identified more patients with osteoarthritis in the younger group than previous studies.\n\nEstimations of burden of disease would differ depending on the methods used and the disability coefficients assigned to the disease. The use of different life expectancy values (West 26 vs GBD 2050) and weights/discounts affects not only the absolute values of DALYs but also the estimations within subgroups13. In addition, the original 1994 WHO method differentiated life expectancy values for men (80 years) and women (82.5 years), penalized the extremes of life ages, and discounted the value of years away in time, reducing the DALYs contributed by men and younger people22–24. The 2015 GBD method tried to correct these differences by using the maximum projected life expectancy for 2050 (91.9 years) without differences between sexes, and discarded age-weighting and time-discounts. The intended effect is to increase the sensitivity of the method to estimate DALYs, especially in the extremes of life. In our study, the 2015 GBD methodology yielded values almost three times higher than the original recommendations, and reduced the gaps between sexes and age groups.\n\nAnother important component when calculating DALYs is the disability weights. The original 1994 WHO methodology recommends using a disability weight of 0.22 for diseases that limit recreation, occupation, education or procreation activities, and 0.40 if a disease limits two or more of these activities. Instead, the 2015 GBD methodology recommends using different weights depending on the severity and location of the disease, giving a disability factor for hip and knee osteoarthritis of 0.165, corresponding to musculoskeletal problems, lower limbs, severe. On the other hand, the MINSA and EsSalud studies have consistently used a 0.28 weight for osteoarthritis regardless of age or sex, because it considers this disease severe enough to seek medical attention. Using the MINSA disability weights instead of the 2015 GBD recommendations increases the absolute values of DALYs by approximately 70%.\n\n\nConclusions\n\nIn the Peruvian social health insurance subsystem, which covers almost 40% of the population, polyosteoarthritis, unspecified osteoarthritis, knee and hip osteoarthritis produced a high burden of DALYs lost, especially among patients over 60 years old and women. The 2015 GBD methodology yields values almost three times higher than the original recommendations, and the disability weights used by MINSA produced estimates 70% higher than using the 2015 GBD weights.\n\n\nData availability\n\nFigshare: db_osteoarthritis_v3.xlsb http://doi.org/10.6084/m9.figshare.1200395717\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nZenodo: culquichicon/gbd-ostheoarthritis: Global burden of disease in osteoarthritis in Peru https://doi.org/10.5281/zenodo.372716716\n\nData are available under the terms of the GNU General Public License version 3 (GPL-3.0).", "appendix": "References\n\nFelson DT: Clinical practice. Osteoarthritis of the knee. N Engl J Med. 2006; 354(8): 841–8. PubMed Abstract \| Publisher Full Text\n\nLane NE: Clinical practice. Osteoarthritis of the Hip. N Engl J Med. 2007; 357(14): 1413–21. PubMed Abstract \| Publisher Full Text\n\nEstudio de carga de enfermedad en el Perú. [Internet]. Ministerio de Salud. 2004. Reference Source\n\nReducir los riesgos y promover una vida sana. [Internet]. World Health Organization. (Informe sobre la salud en el mundo). 2002. Reference Source\n\nMurray CJL, Lopez AD: The Global burden of disease?: a comprehensive assessment of mortality and disability from diseases, injuries, and risk factors in 1990 and projected to 2020. 1996. Reference Source\n\nVelásquez A, Cachay C, Munayco C, et al.: La carga de Enfermedad y Lesiones en el Perú. Lima: Ministerio de Salud; 2009. Report No.: 1.\n\nVelásquez A: La carga de enfermedad y lesiones en el Perú y las prioridades del plan esencial de aseguramiento universal. Rev Peru Med Exp Salud Publica. 2009; 26(2): 222–31. Reference Source\n\nValdez-Huarcaya W, Miranda Monzon J: Estimacion de los años de vida saludables perdidos. Ministerio de Salud. 2014. (Estudio de Carga de enfermedad en el Perú). Reference Source\n\nSeclen Ubillus Y, Depaz Martinez D, Trujillo Navarro J, et al.: Carga de enfermedad y lesiones en EsSalud. Lima: Seguro Social del Peru; 2015.\n\nMezones-Holguín E, Solis-Cóndor R, Benites-Zapata VA, et al.: [Institutional differences in the ineffective access to prescription medication in health care centers in Peru: analysis of the National Survey on User Satisfaction of Health Services (ENSUSALUD 2014)]. Rev Peru Med Exp Salud Publica. 2016; 33(2): 205–14. PubMed Abstract\n\nLlanos RQ, Ramírez RR, Palacios MT, et al.: Health Survey in a Peruvian health system (ENSSA): design, methodology and general results. Rev Saude Publica. 2019; 53: 33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSeuc AH, Domínguez E: Introducción al cálculo de esperanza de vida ajustada por discapacidad. Rev Cuba Hig Epidemiol. 2002; 40: 95–102. Reference Source\n\nWHO methods and data sources for global burden of disease estimates 2000-2015. Geneva: World Health Organization; 2017. Reference Source\n\nSalomon JA: New disability weights for the global burden of disease. Bull World Health Organ. 2010; 88(12): 879. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSalomon JA, Haagsma JA, Davis A, et al.: Disability weights for the Global Burden of Disease 2013 study. Lancet Glob Health. 2015; 3(11): e712–23. Publisher Full Text\n\nCulquichicón C: culquichicon/gbd-ostheoarthritis: Global burden of disease in osteoarthritis in Peru (Version gbd_osteoarthritis). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3727168\n\nCulquichicón C: db_osteoarthritis_v3.xlsb. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12003957.v1\n\nMarshall DA, Vanderby S, Barnabe C, et al.: Estimating the Burden of Osteoarthritis to Plan for the Future. Arthritis Care Res (Hoboken). 2015; 67(10): 1379–86. PubMed Abstract \| Publisher Full Text\n\nFontalba-Navas A, Lucas-Borja ME, Gil-Aguilar V, et al.: Incidence and risk factors for post-traumatic stress disorder in a population affected by a severe flood. Public Health. 2017; 144: 96–102. PubMed Abstract \| Publisher Full Text\n\nCross M, Smith E, Hoy D, et al.: The global burden of hip and knee osteoarthritis: estimates from the global burden of disease 2010 study. Ann Rheum Dis. 2014; 73(7): 1323–30. PubMed Abstract \| Publisher Full Text\n\nValdez-Huarcaya W, Miranda J, Ramos W, et al.: Estimación de la carga de enfermedad por muerte prematura y discapacidad en el Perú. Año 2008. Rev Peru Epidemiol. 2013; 16(2): 1–9. Reference Source\n\nTsuchiya A: Age weighting and time discounting: technical imperative versus social choice. Geneva: World Health Organization; 2002. (Summary measures of population health: concepts, ethics, measurement and applications).\n\nJamison DT, Shahid-Salles SA, Jamison J, et al.: Incorporating Deaths Near the Time of Birth into Estimates of the Global Burden of Disease. En: Lopez AD, Mathers CD, Ezzati M, Jamison DT, Murray CJ, editores. Global Burden of Disease and Risk Factors. Washington (DC): World Bank; 2006. PubMed Abstract\n\nMurray CJ, Ezzati M, Flaxman AD, et al.: GBD 2010: design, definitions, and metrics. Lancet. 2012; 380(9859): 2063–6. steoarthritis incidence among patients from the Peruvian social health insurance system in 2016. PubMed Abstract \| Publisher Full Text" }	[ { "id": "62054", "date": "20 Apr 2020", "name": "Theo Vos", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper reports on a burden of disease analysis of osteoarthritis for Peru. Unfortunately, the researchers do not display adequate knowledge of current or past methods of burden of disease measurement. What they have labelled as their GBD2015 methods is very different from the actual GBD 2015 methods. The prior method is wrongly labelled as WHO method. In the 1990s WHO was not a formal partner with Harvard University and World Bank in producing the earliest round of GBD. I highlight the main errors in the introduction and methods sections below. As these variations to established methods make comparisons impossible, I refrain from making further comments on the results and discussion.\n\nPage 3, second sentence: a global estimate of OA DALYs is referenced to a Peruvian study.\n\nPage 3, third paragraph: Unclear statement ‘…tables based on 1966 data…’.\n“…employed different life expectancy for men and women…”; what is meant is that the standard life expectancy against which years of life lost are estimated had a difference between men and women.\n“..2015 Global Burden of Disease (GBD) study ..”; this is now three iterations of GBD out-of-date …though these general methods still apply.\n“..life expectancy projected to 2050 ..; not correct. I presume it refers to new approach to standard life expectancy which is same for men and women and is based on lowest observed mortality rates in any age group in populations over 5 million.\n\nPage 3, last paragraph: “…We excluded patients whose ICD-10 codes for osteoarthritis were registered.\n\n\"during hospitalization and patients who were previously treated for osteoarthritis….”; how?\n\nPage 3/4: “…Sample size calculation estimated that a minimum of 20,000 records were needed to find differences of at least four DALYs/1000 people between subgroups with 80% power and 95% significance.”; no reason to sample when using medical claims data.\n\nPage 4, second paragraph: “Covariates…”, better to use term ‘Variables…’.\n\nPage 4: ‘The original WHO burden of disease study from 1994 ..’; This was a study by Harvard University for World Bank, not WHO.\n\nPage 4: ‘The second method, employed in the 2015 GBD study, uses the maximum worldwide life expectancy projected to 2050…’ Incorrect statement; see comment above.\n\nPage 4: ‘…the YLL was calculated as the life expectancy at the time of death using the two methods described above12,13…’; the two references are both for methods prior to GBD 2010.\n\nPage 4: ‘..Given that osteoarthritis is a chronic condition that lasts until death, the average duration of the illness was considered as the life expectancy at the time of initial diagnosis. For this estimation, we used the two methods previously described12,13…’; same comment as previous, that the two references refer to methods prior to GBD2010 only. What is not stated is a big methods change in non-fatal estimation from GBD2010 onwards estimating YLDs based on prevalence and not incidence. Thus, the authors incorrectly describe GBD2015 methods and their results, therefore, are not comparable to those of GBD 2015 (or any iteration after GBD 2010).\n\nPage 5: ‘……Two disability weights were used: 0.165, which is the value employed by the 2015 GBD study for severe musculoskeletal diseases of lower limbs14,15..’; question 1: why this selective choice of applying the more severe of DWs to all cases?; question 2: where did the Peruvian DW come from? What method was used?\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? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "62049", "date": "04 Jun 2020", "name": "Mario Valladares-Garrido", "expertise": [ "Reviewer Expertise Global health", "internal medicine", "mental health." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall, the findings match with Peruvian estimates of the global burden of disease (GBD) in osteoarthritis done by the Peruvian Ministry of Health and the Social Health Insurance in their previous reports over the years.\nA study strength is that the authors report both methodologies (1994 and 2015) for the global burden of disease estimations, in that sense, they address a gap of knowledge between Peruvian reports.\nThis study contributes to Peruvian GBD estimations especially in chronic non-communicable diseases with great impacts on elderly people.\n\nIs the work 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": "64466", "date": "22 Jun 2020", "name": "Reneé Pereyra-Elías", "expertise": [ "Reviewer Expertise Social epidemiology", "mental health", "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\nThis manuscript covers an important topic, that has not been explored much in Peru. Due to its relevance and the population included in the study (all those insured by EsSalud, that provides insurance to a significant fraction -about 1/3- of the Peruvian population), I consider it is worth publishing. However, it is necessary to review some important points.\n\nI think the main aspect to review would be the construction of the 'burden of disease' variables. I am not an expert in these metrics. I highly recommend reading and considering the detailed suggestions of reviewer Prof. Theo Vos, who is a world expert in the field.\n\nMinor revisions:\nSample size: The authors state that they were trying to detect differences between subgroups? Which subgroups? Knee and hip osteoarthritis? Males and females? Age groups? There are no hypothesis tests reported throughout the manuscript. Please clarify that.\n\nIt would be interesting to see some potential explanations for the regional differences between the burden of disease. Is it due to the timing of diagnosis? Is it due to the characteristics of these populations?\n\nThe manuscript might benefit from an English-language edit.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-238
https://f1000research.com/articles/9-988/v1	14 Aug 20	{ "type": "Research Article", "title": "Incidental findings on computed tomography coronary angiography and its impact on respiratory services in a United Kingdom district general hospital", "authors": [ "Bikash Gurung", "Finnian D. Lesser", "Ellis James", "Kabali Nandakumar", "Bikash Gurung", "Ellis James", "Kabali Nandakumar" ], "abstract": "Background: Computed tomography coronary angiography is used to assess for coronary artery disease but can also pick up non-cardiac pathology. Previous studies have assessed the frequency of non-cardiac pathology. We investigated the non-cardiac findings and resulting follow up in a District General Hospital. Methods: All computed tomography coronary angiography scans for 1 year were retrospectively collected. Basic demographics and the non-cardiac findings were recorded from electronic health records. The significant respiratory findings and the respiratory follow up of these non-cardiac findings were recorded. Results: A total of 503 scans were carried out in one year. Of these scans, 24% had non cardiac findings present. Older patients were more likely to have non cardiac findings. The most common non cardiac findings were lung nodules, emphysema and hiatus hernias. Significant respiratory findings were present in 35 cases, which generated 24 episodes of respiratory follow up. Some patients who met criteria for follow up had not been referred. Conclusions: Non cardiac findings are common on computed tomography coronary angiography and in our hospital these findings led to significant follow up in respiratory services.", "keywords": [ "computed tomography coronary angiogram", "incidental findings", "lung nodule" ], "content": "Introduction\n\nComputed tomography coronary angiography (CTCA) is used to assess for coronary artery disease in patients with chest pain and in preprocedural planning. In the United Kingdom the National Institute for Health and Care Excellence (NICE) guidelines 2016 have recommended CTCA as the first-choice imaging modality in patients with chest pain1. Aside from imaging the coronary arteries, CTCA will also image surrounding structures and can pick up non-cardiac findings (NCF). Some of the NCF will need follow up and further investigation, which has cost and service delivery implications2.\n\nThe NCF of CTCA have been previously investigated in prospective and retrospective cohort studies with greatly varying proportions of incidental finding in scans2–15. A systematic review of all forms of cardiac CT found NCF in 7%-100% of studies included16. The most common findings in previous studies have been respiratory, in particular lung nodules.\n\nThe British thoracic society guidelines and the Fleischner society have guidelines of the follow up of lung nodules17,18.\n\nIn this study of a district general hospital we assessed the frequency of NCF on CTCA, their significance and the follow up requested. The hospital provides secondary care services for cardiology, respiratory, gastroenterology and orthopaedics among other services.\n\n\nMethods\n\nEthical approval for data collection and analysis was received from our institution’s clinical effectiveness team and all patient data has been anonymised. As the data was retrospectively collected and did not affect the included patients care no further ethical approval was required.\n\nAll CTCA performed for one year (2018) were collected retrospectively. Data were reviewed on electronic health records with demographics of patient’s age and sex recorded in an Excel spreadsheet. The CTCA’s had been performed on a Toshiba Acquillion One using the standard protocol and the images reported by consultant radiologists. NCF were recorded and divided by speciality. Patients with respiratory NCF had their notes reviewed for significance of findings, initial referral and follow up. Findings were assessed as significant if they required further investigation, follow up or treatment. For lung nodules significance was determined by whether they met criteria for further follow up according to British Thoracic Society Guidelines18. If a significant finding had not been followed up the primary care provider for the patient involved was informed.\n\nStatistical analysis was performed using Jamovi (version 1.2.22)19. Normally distributed data are presented as means and standard deviations. Statistical significance was assessed by t-test or chi squared test, as appropriate. A p value <0.05 was deemed significant.\n\n\nResults\n\nA total of 503 CTCA were requested in 12 months with nearly all being requested by cardiology and 3 by primary care. There were 284 scans on females and 219 scans on males. The average age of the patients was 60.\n\nNCF were identified in 120 (24%). NCF were more frequent in older patients with statistical significance. For gender there was no statistically significant difference in likelihood to have NCF (Table 1). Details of all CTCA and resultant NCF are available as Underlying data20.\n\nOf the NCF identified, 95 were respiratory with the majority being lung nodules (Table 2). The respiratory NCF were judged to be significant in 35 cases (7% of total scans). Other NCF were found in gastroenterology, orthopaedic and endocrine specialties which we did not assess for significance.\n\nOnward referral for imaging (all CT Chest) was requested in 18 patients with results followed up by the respiratory team (Table 3). In addition to this, 5 patients were reviewed in the respiratory clinic without further imaging and 1 in a multidisciplinary team meeting.\n\nThere were 16 patients who had significant respiratory findings not referred for secondary care follow up, of these 9 were emphysema or parenchymal changes, 5 were nodules and 1 was a pleural effusion.\n\n\nDiscussion\n\nNCF on CTCA are common, but the majority of these are not significant. If the use of CTCA increases, this will lead to significant downstream effects on other specialties, especially respiratory. In our hospital, one year’s worth of CTCA led to 24 respiratory follow ups in terms of imaging and clinic appointments. Even non-significant findings may lead to referrals and investigations if the ordering clinician is unsure of the current guidelines.\n\nOther similar studies show a large variability in proportion of NCF in CTCA, likely due to variability in study populations, equipment and protocols used. Our results are similar to the most recent large UK dataset from the SCOT HEART trial with NCF more frequent as age increases and the most common findings being lung nodules, emphysema and hiatus hernia2. These additional data from a single district general hospital should inform other healthcare providers of possible service provision consequences from the use of CTCA.\n\nWe identified several patients who would have met criteria for follow up with lung nodules but did not receive this follow up. For some of the NCF such as emphysema and hiatus hernia the patients primary care practitioner will be the correct route of follow up and so they should be informed of the result. As these patients are all symptomatic enough to warrant a CTCA optimising their treatment for NCF seems prudent particularly as these may be the underlying cause of the symptoms. Not correctly following up lung nodules can have significant implications for the patient and in our data set some patients were not followed up according to guidelines.\n\nCardiologists may not be familiar with the follow up of NCF and so should be cautious when interpreting scan reports. As NCF are common in CTCA a standardised method of reviewing scans and referring findings could be considered with the aim to reduce unnecessary follow up requests and missed significant findings.\n\n\nConclusion\n\nNCF are frequent on CTCA and will lead to downstream follow up which will have implications in terms of cost, service provision and patient’s time.\n\n\nData availability\n\nFigshare: Data from: Incidental findings on computed tomography coronary angiography and its impact on respiratory services in a United Kingdom district general hospital. https://doi.org/10.6084/m9.figshare.12600056.v220.\n\nThis project contains details of each computed tomography coronary angiography procedure performed, including a description of the non-cardiac findings, where applicable. To anonymise the data patient age and date of birth is not included.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nOverview \| Recent-onset chest pain of suspected cardiac origin: assessment and diagnosis \| Guidance \| NICE. NICE; [cited 2020 Jun 18]. Reference Source\n\nWilliams MC, Hunter A, Shah ASV, et al.: Impact of noncardiac findings in patients undergoing CT coronary angiography: a substudy of the Scottish computed tomography of the heart (SCOT-HEART) trial. Eur Radiol. 2018; 28(6): 2639–46. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaller S, Kaiser C, Buser P, et al.: Coronary Artery Imaging with Contrast-Enhanced MDCT: Extracardiac Findings. AJR Am J Roentgenol. 2006; 187(1): 105–10. PubMed Abstract \| Publisher Full Text\n\nKarius P, Lembcke A, Sokolowski FC, et al.: Extracardiac findings on coronary computed tomography angiography in patients without significant coronary artery disease. Eur Radiol. 2019; 29(4): 1714–23. PubMed Abstract \| Publisher Full Text\n\nJohnson KM, Dennis JM, Dowe DA: Extracardiac findings on coronary CT angiograms: Limited versus complete image review. AJR Am J Roentgenol. 2010; 195(1): 143–8. PubMed Abstract \| Publisher Full Text\n\nKawano Y, Tamura A, Goto Y, et al.: Incidental detection of cancers and other non-cardiac abnormalities on coronary multislice computed tomography. Am J Cardiol. 2007; 99(11): 1608–9. PubMed Abstract \| Publisher Full Text\n\nBoldeanu I, Perreault Bishop J, Nepveu S, et al.: Incidental findings in CT imaging of coronary artery bypass grafts: results from a Canadian multicenter prospective cohort. BMC Res Notes. 2018; 11(1): 72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLazoura O, Vassiou K, Kanavou T, et al.: Incidental Non-Cardiac Findings of a Coronary Angiography with a 128-Slice Multi-Detector CT Scanner: Should We Only Concentrate on the Heart? Korean J Radiol. 2010; 11(1): 60–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOnuma Y, Tanabe K, Nakazawa G, et al.: Noncardiac Findings in Cardiac Imaging With Multidetector Computed Tomography. J Am Coll Cardiol. 2006; 48(2): 402–6. PubMed Abstract \| Publisher Full Text\n\nMachaalany J, Yam Y, Ruddy TD, et al.: Potential clinical and economic consequences of noncardiac incidental findings on cardiac computed tomography. J Am Coll Cardiol. 2009; 54(16): 1533–41. PubMed Abstract \| Publisher Full Text\n\nCho JH, Park JS, Shin DG, et al.: Prevalence of extracardiac findings in the evaluation of ischemic heart disease by multidetector computed tomography. J Geriatr Cardiol. 2013; 10(3): 242–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChia PL, Kaw G, Wansaicheong G, et al.: Prevalence of non-cardiac findings in a large series of patients undergoing cardiac multi-detector computed tomography scans. Int J Cardiovasc Imaging. 2009; 25(5): 537–43. PubMed Abstract \| Publisher Full Text\n\nLaw YM, Huang J, Chen K, et al.: Prevalence of significant extracoronary findings on multislice CT coronary angiography examinations and coronary artery calcium scoring examinations. J Med Imaging Radiat Oncol. 2008; 52(1): 49–56. PubMed Abstract \| Publisher Full Text\n\nGil BN, Ran K, Tamar G, et al.: Prevalence of Significant Noncardiac Findings on Coronary Multidetector Computed Tomography Angiography in Asymptomatic Patients. J Comput Assist Tomogr. 2007; 31(1): 1–4. PubMed Abstract \| Publisher Full Text\n\nLehman SJ, Abbara S, Cury RC, et al.: Significance of cardiac computed tomography incidental findings in acute chest pain. Am J Med. 2009; 122(6): 543–9. PubMed Abstract \| Publisher Full Text\n\nKay FU, Canan A, Abbara S: Common Incidental Findings on Cardiac CT: a Systematic Review. Curr Cardiovasc Imaging Rep. 2019; 12(6): 21. Publisher Full Text\n\nMacMahon H, Naidich DP, Goo JM, et al.: Guidelines for Management of Incidental Pulmonary Nodules Detected on CT Images: From the Fleischner Society 2017. Radiology. 2017; 284(1): 228–43. PubMed Abstract \| Publisher Full Text\n\nCallister MEJ, Baldwin DR, Akram AR, et al.: British Thoracic Society guidelines for the investigation and management of pulmonary nodules. Thorax. 2015; 70 Suppl 2: ii1–ii54. PubMed Abstract \| Publisher Full Text\n\nThe jamovi project. Jamovi. 2020. Reference Source\n\nGurung B, Lesser FD, James E, et al.: Data from: Incidental findings on computed tomography coronary angiography and its impact on respiratory services in a United Kingdom district general hospital. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12600056.v2" }	[ { "id": "96899", "date": "08 Feb 2022", "name": "Mohammad Kermani-Alghoraishi", "expertise": [ "Reviewer Expertise Cardiovascular Medicine" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI reviewed this paper regarding to incidental finding in patients underwent CCTA.\n\nThis is good idea, but the methodology and statistics are weak. Also, this title has not enough novelty and it is practical locally.\nOne of the most important limitations of this study is that CCTA technical approach is different with lung and mediastinal CT approaches and it can not conclude about the lung diseases accurately.\nAlso, it was better that authors analysis the relation between the incidental findings with CCTA results as well as demographic 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? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-988
https://f1000research.com/articles/9-978/v1	12 Aug 20	{ "type": "Research Article", "title": "Kannada akshara knowledge in primary school children: measurement of accuracy and reaction time using a cross-sectional study design", "authors": [ "Mysore Nanda Kumar Usha", "Malavika Anakkathil Anil", "Shwetha Prabhu", "Jayashree S. Bhat", "Somashekara Haralakatta Shivananjappa", "Mysore Nanda Kumar Usha", "Malavika Anakkathil Anil", "Shwetha Prabhu", "Jayashree S. Bhat" ], "abstract": "Background: Reading acquisition varies between languages, as languages differ in terms of phonology and orthography. Orthographic knowledge is demonstrated to be crucial in literacy acquisition in most orthographies. The literature on acquisition of orthographic knowledge has focused more on alphabetic orthographies and less is understood in alphasyllabary Kannada language. The present study aimed to understand the akshara knowledge acquisition by measuring akshara identification accuracy and reaction time in typically developing Kannada medium primary school children. Methods: The study consisted of 315 typically developing children, 45 each from Grade I through Grade VII between the age range of 5 years 6 months to 12 years 6 months. The children were assessed for akshara identification accuracy and reaction time using a representative sample of 67 akshara selected at four different levels of complexity: vowels in primary form, consonant with inherent vowels, consonant with vowel diacritics, and consonant clusters. The mean performance was compared between the groups using one-way ANOVA with post-hoc Bonferroni test. Results: One-way ANOVA revealed significant main effect (p≤0.05) of Grade on akshara identification accuracy and reaction time. The post-hoc Bonferroni test revealed that the mean akshara identification accuracy improved significantly (p≤0.05) from Grade I to Grade V and reached a plateau at Grade VI. The reaction time significantly reduced from Grade I to Grade IV and there was no significant change beyond Grade V.\n\nConclusion: The children learning to read alphasyllabary Kannada gain mastery over the majority of aksharas during the initial years of formal schooling, which develops completely by Grade VI. The automaticity in naming akshara develops gradually and reaches a plateau by Grade IV. The present findings indicate that children acquire automaticity in naming akshara early, while the akshara knowledge continues to develop.", "keywords": [ "Akshara knowledge", "Alphasyllabary", "Akshara identification accuracy", "Reaction Time" ], "content": "Introduction\n\nReading is a process of developing a sense of written material through systematic mapping of phonemes to the corresponding grapheme. Language-specific phonological and orthographic knowledge is crucial for successful word decoding. Orthographic knowledge is the information that is stored in memory, which dictates the rules to represent the spoken language in the written form (Apel, 2011). The role of orthographic knowledge is crucial in literacy acquisition (e.g., Apel et al., 2006; Castles & Coltheart, 2004). The studies conducted in alphabetical languages have reported that orthographic knowledge acts as the strongest predictor of reading success in children (Bowey, 2005; Caravolas et al., 2013; Hulme et al., 2012; Seymour, 2005). However, there are prevailing arguments about the generalization of theoretical models explaining the reading process and other research findings from alphabetic writing systems to children learning to read in other writing systems (Karanth, 2003; Nag, 2007). Studies in alphasyllabaries have shown that the developmental pathway of reading is different from children acquiring an alphabetic writing system (Nag, 2007; Nag & Snowling, 2011; Nag & Snowling, 2012). Alphasyllabaries are phonologically and orthographically different from alphabetic writing systems and share features of both alphabetic and syllabic writing systems. Previous studies have illustrated clear differences between alphabetic and alphasyllabaries languages (e.g., Nag, 2007; Nag, 2014; Tiwari et al., 2011). Alphasyllabaries represent sounds at the level of the syllable called akshara, but have distinct features to indicate sub-syllabic information (Bright, 1999). These differences explain the variability in the acquisition of orthographic knowledge across orthographies. For example, Seymour et al. (2003) reported that children acquired orthographic knowledge by the end of Grade I in alphabetical language such as English, whereas Shu (2003) reported it to be beyond Grade VI in Chinese.\n\nPrevious attempts have examined akshara knowledge in alphasyllabary languages, such as Kannada and Malayalam. Nag (2007) investigated children’s acquisition of orthographic knowledge in Kannada, a south Indian alphasyllabary language. The study included a group of 374 primary school children between the age range of 5 to 10 years, studying in regular schools with Kannada language as the medium of instruction. The children were assessed for akshara knowledge twice. The first assessment was done when they were Grade I, II, and III. The follow-up assessment was done after 15 months when the children moved to subsequent Grades. A Syllabograph Recognition Test (LAB, 2003) was conducted, which consists of 20 Kannada akshara, in which one akshara was a primary vowel, eight aksharas of consonants with inherent vowels, five aksharas of consonants with vowel diacritics, and six aksharas of consonant clusters. The results revealed a significant main effect of time of testing and Grade level with a significant interaction between these variables on akshara knowledge. Akshara mastery was found to continue into elementary school with an accuracy of around 80% during the second assessment. The study noted the differential trend for individual akshara types; consonants with inherent vowels were mastered by the end of Grade I, consonants with vowels diacritics with ligatures and consonant clusters were only partially mastered by Grade II, and this extended beyond Grade IV with a substantial number of children showing less than 50% accuracy even at Grade IV. These findings indicated that orthographic knowledge in Kannada was determined by the orthographic features of akshara.\n\nAnother study was conducted by Tiwari et al. (2011) in Malayalam, which is another alphasyllabary language. This study investigated akshara knowledge in Grade III children learning to read alphasyllabary. The study included 80 children between the age range of 7–8 years from two Malayalam medium schools in Kerala. The stimuli were similar to the Nag (2007) study, which included 17 Malayalam aksharas, such as a primary form of vowels, consonants with inherent vowels, consonants with vowels diacritics, and consonant clusters. The aksharas was presented in the flashcards one at a time, and the children were asked to name them. The results showed a differential pace of mastery of various akshara types in Malayalam alphasyllabary. Grade III children mastered vowels in primary form and consonants with inherent vowels. Consonants with vowel diacritics showed partial dispersion, whereas the consonant cluster showed extreme dispersion. They also reported that consonant with vowel diacritics was mastered much better than consonant clusters.\n\nNag (2007) and Tiwari et al. (2011) have indicated that the acquisition of akshara knowledge continues beyond Grade III in Malayalam and Grade IV in Kannada. However, there is a paucity of data beyond Grade IV in alphasyllabary languages, specifically in Kannada. Additionally, the studies in alphasyllabary have used a limited sample of aksharas out of a large registry, thus limiting the generalization of the findings. More importantly, the studies in alphasyllabaries have determined mastery by assessing the accuracy of akshara identification. However, fluent reading requires the speed with which the individual aksharas are retrieved from long-term memory. Hence, the speed with which akshara naming can be determined by measuring reaction time for individual aksharas. The speed along with accuracy in recognizing aksharas enables children to decode the words faster, thereby enhancing fluency in reading, resulting in improved comprehension. Hence the present study aimed to develop a comprehensive understanding of akshara knowledge acquisition by measuring both accuracy and reaction time in typically developing Kannada medium primary school children from Grade I to Grade VII within the age range of 5 years 6 months to 12 years 6 months.\n\n\nMethods\n\nThe present research incorporated cross sectional study design with non-random convenient sampling method to recruit the participants. The present research was approved by the Institutional Ethic Committee of Kasturba Medical College, Mangalore, India (IEC KMC MLR 11-18/472). The study was conducted between December 2018 and February 2020. The children from five government schools belonging to urban Mangalore of Dakshina Kannada district affiliated to the Karnataka state board with Kannada as the medium of instruction served as participants. Permission from the school administration and written consent from the parents of the participants was obtained before initiating the study.\n\nThe study consisted of 315 children, 45 each from Grade I through Grade VII between the age range of 5 years 6 months to 12 years 6 months. The children were divided into seven equal groups based on the Grade and respective age range, as shown in Table 1. The sample size was calculated using the formula n= 2(Zα+Zβ)² σ² / d2 Zα-1.96 at 95% confidence level, Zβ-0.84 at 80% power, based on the study done by Nag (2007). The participants were selected based on the following selection criteria:\n\nInclusion criteria. Children who (a) fit into the age and Grade criteria; (b) belong to regular government Kannada medium school; (c) have either Tulu, Konkani or Beary as a native language (L1); (d) are reported to have average and above-average scholastic performance by the class teachers; and (e) belong to families of middle socio-economic status.\n\nExclusion criteria. Children with (a) significant speech, language, hearing, developmental, intellectual, and neurological disorders, according to the WHO ten-question disability screening checklist (Singhi et al., 2007); (b) history of class retentions; (c) poor academic performance according to class teachers report; (d) poor attendance according to school records; and (e) a history of change in the medium of instructions.\n\nKannada akshara knowledge was assessed by measuring akshara identification accuracy and reaction time using a representative sample of a Kannada orthographic inventory. A total of 67 aksharas at four different levels of complexity were selected (Table 2).\n\nTo facilitate the akshara naming task, the aksharas from the representative sample were initially typed in Kannada orthography with black colored font in a Microsoft PowerPoint (PPT) using Nudi software v5.0. A constant font type ‘Nirmal Ul’ and font size 250 were maintained. The PPTs were converted as a JPEG image to create an individual akshara image having uniform dimension.\n\nThe aksharas were presented visually through a laptop installed with licensed version of Paradigm Experiment software v2.5.0.68, which facilitates data acquisition on akshara naming accuracy as well as reaction time. (DmDX 5.1 software is a freely available alternative that could be used.) The Paradigm Experiment software was programmed to present the individual aksharas randomly but in a fixed order for every participant with 3000ms onscreen stimulus duration and 2500ms inter-stimuli interval. The system was connected to a microphone, which recorded the participant’s vocal responses and provided the reaction time automatically in an Excel document. The reaction time was measured in milliseconds, and is defined as the total time elapsed between the presentation of akshara and the onset of the correct naming. Once the responses were recorded, the accuracy of the correct identification of aksharas was checked manually by listening to the response offline and noting down the corresponding reaction time for further statistical analysis. During the analysis, every accurate naming of aksharas was scored ‘1’ and ‘0’ for inaccurate naming. The reaction time was considered only for the correct responses. The calculated accuracy of the response and the corresponding reaction time were tabulated and subjected to statistical analysis.\n\nThe participants were made to sit comfortably in front of the laptop in a classroom with relatively low ambient noise with adequate illumination. The laptop was connected headset with microphone positioned at participant’s eye-level with a constant distance of 1 meter. The participants from each group were tested individually. The participants were instructed to name the aksharas as quickly and correctly as possible. All the participants were familiarized with the task through practice trials before the actual testing.\n\nStatistical analysis was carried out using SPSS software version 17.0. Initially, the raw data were summarized in terms of mean, standard deviation, and range of scores using descriptive statistics. Later, the performance was compared across the Grades using one-way ANOVA with post-hoc pair-wise comparison using the Bonferroni test. Later step-wise linear multiple regression analysis was carried out to investigate the independent contribution of akshara identification accuracy and reaction time in word decoding skills. A statistical significance of p<0.05 was considered significant in the present study.\n\n\nResults\n\nFigure 1 and Figure 2 show the mean and SD for akshara identification accuracy and reaction time across the Grades, respectively.\n\nThe result of one-way ANOVA revealed a significant main effect of Grades on akshara identification accuracy [F (6, 308) = 1687.057, p=0.00] and reaction time [F (6, 308) = 69.84, p= 0.00]. Subsequently, a post-hoc pair-wise multiple group comparison was performed using the Bonferroni test, which revealed that the mean performance of Grade I on akshara identification accuracy was significantly lower than higher Grades (p≤0.05). This result was the same for Grades II, III, IV and V in comparison to higher Grades (p≤0.05). However, there was no significant difference between Grade VI and Grade VII (p≥0.05).\n\nThe post-hoc pair-wise multiple group comparison of mean reaction time using Bonferroni test failed to reveal any significant difference (p≥0.05) in mean performance between Grade I and II, but Grade I children took significantly more time (p≤0.05) in naming aksharas than all higher Grades. This result was the same for Grades II and III in comparison to all higher Grades (p≤0.05). However, there was no statistically significant difference between Grade IV and Grade V, but the arithmetical mean showed Grade IV took a longer time than Grade V. The performance of Grade IV was significantly lower than Grades VI and VII (p≤0.05). Similarly, there was no significant difference (p≥0.05) noted in the mean performance between Grades V and VII, but the mean performance of Grade V was significantly lower than Grade VII (p≤0.05). There was no significant difference noted (p≥0.05) in the mean performance between Grades VI and VII, which indicated that children from Grades I to IV took significantly greater time to identify the akshara compared to Grades VI and VII who took substantially less time.\n\n\nDiscussion\n\nThe results of one-way ANOVA revealed that accuracy in identifying akshara improves with Grade. On post-hoc pair-wise multiple group comparison, it was observed that the mean performance of children in identifying Kannada aksharas improved significantly from Grade I through to Grade V and reached a plateau at Grade VI, indicating a steady improvement in children’s akshara identification scores. The results showed a developmental trajectory, where children mastered every level of representative aksharas gradually in each Grade, and completely acquired all levels of representative aksharas by Grade VI. These findings are in agreement with results obtained by Nag (2007), who reported that the acquisition of akshara knowledge goes beyond ten years of age (Grade IV). In alphabetical languages, like English, children are reported to master letter knowledge by Grade I (Seymour et al., 2003) due to the lower number of graphemes compared to alphasyllabary languages, which consists of an extensive number of graphemes and also requires the mastery of a large group of orthographic register and their ligaturing rules (Nag, 2007). In alphabetic knowledge, the acquisition of letters of the alphabet takes place in a gradual manner, i.e. children learn the letters of the alphabet on the letter by letter basis, rather than learning the entire set at once. Therefore, young children are introduced initially to consonants followed by vowels, as consonants are more consistent in their sounds and hence can be quickly learned compared to vowels (Strickland, 1998). The gradual, steady improvement in the identification of akshara can be attributed to the manner in which aksharas are taught in schools. Generally, aksharas are taught to children in a hierarchical manner where they are introduced to the aksharas based on the level of complexity, i.e. initially, children are introduced to primary vowels and consonant with inherent vowels, followed by consonants with vowel diacritics, and consonant clusters (Karanth & Prakash, 1996).\n\nThe findings of reaction time measurement from the present study revealed that the children from Grade I to IV took a significantly greater time to identify aksharas compared to Grades V, VI and VII, who took significantly less time. This implies that younger children took a significantly longer time in identifying aksharas compared to older groups. The reduction in reaction time can be described as developmental changes in the speed of information processing (Kail & Salthouse, 1994; Kail, 2008). Studies report that increases in the speed of processing are correlated with age-related improvements in performance on a variety of cognitive tasks, including reading, memory, solving arithmetic problems, finding their way, and reasoning (Kail, 2004; Kail, 2008). Even though there was an increase with Grades, the performance of reaction time reached a plateau at Grade IV, and there was no significant difference noted in time taken to name aksharas above Grade IV. The occurrence of plateau in reaction time can be attributed to familiarity of the curricular material to which children are exposed frequently during primary school years. For example, most of the text introduced to Grade I children included words which are formed by primary vowels and consonant with inherent vowels, while in older grades, children are introduced with the words with diacritic markers and clusters, thus making them more familiar to those levels of complexity. As a result, children are more familiar with the aksharas which makes naming speech reach a constant level, but exploring and learning new aksharas continues across grades. Thus, automaticity in naming aksharas develops prior to akshara identification accuracy, which continues to develop above Grade IV and is mastered by Grade VI.\n\n\nConclusion\n\nChildren learning to read alphasyllabary Kannada language gain mastery over majority of the aksharas during the first five years of formal schooling and this develops completely by Grade VI. The automaticity in naming akshara develops gradually and reaches a plateau by Grade IV itself. The present findings indicate that children acquire automaticity in naming akshara early, whereas the akshara knowledge continues to develop.\n\n\nData availability\n\nHarvard Dataverse: Akshara knowledge in Kannada language, https://doi.org/10.7910/DVN/7TJIMS (Shivananjappa, 2020).\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nApel K: What is orthographic knowledge? Lang Speech Hear Serv Sch. 2011; 42(4): 592–603. PubMed Abstract \| Publisher Full Text\n\nApel K, Wolter JA, Masterson JJ: Effects of phonotactic and orthotactic probabilities during fast mapping on 5-year-olds’ learning to spell. Deve Neuropsychol. 2006; 29(1): 21–42. PubMed Abstract \| Publisher Full Text\n\nBowey JA: Predicting individual differences in learning to read. The Science of Reading: A handbook. 2005; 155–172. 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J Res Read. 2007; 30(1): 7–22. Publisher Full Text\n\nNag S: Alphabetism and the science of reading: From the perspective of the akshara languages. Front Psychol. 2014; 5: 866. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNag S, Snowling MJ: Cognitive profiles of poor readers of Kannada. Read Writ. 2011; 24(6): 657–676. Publisher Full Text\n\nNag S, Snowling MJ: Reading in an Alphasyllabary: Implications for a language universal theory of learning to read. Sci Stud Read. 2012; 16(5): 404–423. Publisher Full Text\n\nSeymour PH, Aro M, Erskine JM, et al.: Foundation literacy acquisition in European orthographies. Br J Psychol. 2003; 94(Pt 2): 143–74. PubMed Abstract \| Publisher Full Text\n\nSeymour PH: Early reading development in European Orthographies. In The science of reading: A handbook, Blackwell Publishing, 2005; 296–315. Publisher Full Text\n\nShivananjappa SH: Akshara knowledge in Kannada language Harvard Dataverse, V1 UNF:6:UPnjvNxCEaLomCxG6X3wtQ== [fileUNF]. 2020. http://www.doi.org/10.7910/DVN/7TJIMS\n\nShu H: Chinese writing system and learning to read. Int J Psychol. 2003; 38(5): 274–285. Publisher Full Text\n\nSinghi P, Kumar M, Malhi P, et al.: Utility of the WHO Ten Questions screen for disability detection in a rural community- the north Indian experience. J Trop Pediatr. 2007; 53(6): 383–387. PubMed Abstract \| Publisher Full Text\n\nStrickland DS: Teaching Phonics Today: A Primer for Educators. Order Department, International Reading Association, 800 Barksdale Road, PO box 8139, Newark, DE 19714-8139. 1998. Reference Source\n\nTiwari S, Nair R, Krishnan G: A preliminary investigation of akshara knowledge in the Malayalam alphasyllabary: Extension of Nag’s (2007) study. Writ Syst Res. 2011; 3(2): 145–151. Publisher Full Text" }	[ { "id": "69436", "date": "03 Sep 2020", "name": "Pradeep Yuvaraj", "expertise": [ "Reviewer Expertise Neuroaudiology", "Electrophysiology", "Adult communication disorders" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article begins with a well writen introduction where in the justification for the present study is noted. The introduction though states that there are literature available which suggests that the reaction time gets better with age, the present study aims to find the plateau for reaction time. The research question is clear and unambiguous.\nThe method section describes the test material, inclusion and exclusion criteria and the mode of stimulus presentation comprehensively.\n\nThe statistical analysis for the group and between group are well described in the results chapter. The demographics are well represented graphically.\nThe discussion derives the inference from the available literature with proper justification for the results from the present study. However the results should have thrown up little more information on neurobiology for processing with age.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "72285", "date": "02 Oct 2020", "name": "Sangamanatha Ankmnal-Veeranna", "expertise": [ "Reviewer Expertise Development and electrophysiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 examines the akshara knowledge in primary school children. The akshara knowledge acquisition was examined by measuring the akshara identification accuracy and reaction time. Study participants included typically developing children between the ages of 6 to 12 years. The introduction is well written. The authors have briefly explained about the alphabetic and alphasyllabaries languages. Authors have also reported previous studies that were carried out to examine the development of akshara knowledge in Kannada and Malayalam language. The authors have discussed the findings of their study.\n\nThe following are my comments regarding the manuscript.\nIntroduction: I have some questions regarding the justification for the need for the study. Why do authors think that it is important to examine the acquisition of akshara knowledge beyond grade IV?\nMethod: How long was the experiment? Did children receive feedback for their responses? The authors have mentioned that scoring was carried out by listening to the recorded sample. Who carried out the scoring?\nStatistical Analysis: Authors have mentioned that they carried out the multiple linear regression analysis on the data. But I do not see those analyses in the result section. Is there any association between the reaction time and the accuracy of akshara identification?\nDiscussion: Why the knowledge of maturation of akshara identification is important? Are their clinical implications?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-978
https://f1000research.com/articles/9-977/v1	12 Aug 20	{ "type": "Case Report", "title": "Case Report: Complex parapneumonic effusion and bacteremia secondary to Granulicatella adiacens in immunocompetent host", "authors": [ "Ajita Acharya", "Zeeshan Sattar", "Ammar Ahmad", "Farhan Sattar", "Aleksey Fiksman", "Ajita Acharya", "Ammar Ahmad", "Farhan Sattar", "Aleksey Fiksman" ], "abstract": "Granulicatella adiacens is a fastidious Gram-positive streptococcus that is linked with causing a variety of infections in immunocompromised hosts. These include endocarditis, bacteremia, osteomyelitis, and spontaneous bacterial peritonitis. We present a rare incidence of this bacterium causing a complex parapneumonic effusion in an immunocompetent host. An effective antibiotic regimen for treating infections caused by this bacterium includes ampicillin plus penicillin plus gentamicin but resistance to various classes of antibiotics has been reported. In conclusion, this organism can affect different systems in immunocompromised as well as immunocompetent hosts, and a high index of suspicion is required for its diagnosis.", "keywords": [ "granulicatella", "adiacens", "parapneumonic", "pneumonia" ], "content": "Introduction\n\nGranulicatella adiacens is a member of the Granulicatella genus of the fastidious nutritionally variant group of the Gram-positive streptococci. The most common clinical syndromes associated with this organism are Endocarditis and Bacteremia, but cases of vertebral osteomyelitis and spontaneous bacterial peritonitis have also been reported1,2. To the best of our knowledge, we are presenting the first case of complex parapneumonic effusion with bacteremia secondary to G. adiacens.\n\n\nCase presentation\n\nA 56 year old male with a history of seizure disorder presented to the Emergency Department with shortness of breath for three days. He also reported productive cough with yellow sputum, right sided chest pain, anorexia, night sweats, fatigue and fever for the same duration. The patient denied any hemoptysis, chills, rigors, leg swelling and recent travel, but had an episode of upper respiratory tract infection three months ago due to sick contact. The patient’s past surgery history was significant for subdural hematoma treated with frontal craniotomy 20 years ago. In addition, the patient used to take dilantin for his seizure disorder, but stopped taking this medication a long time ago. The patient’s mother had a history of breast cancer. Th patient is a smoker for 38 years, and smoked half a pack of cigarettes a day. He is a chronic alcoholic and had drunk 3–4 beers every day for many years, but denies any illicit drug use. He lives with his family.\n\nOn admission, he was febrile to 100.6 F, tachycardic to 150 bpm, hypertensive to 157/93 and saturating at 91% in room air. In the general examination, the patient was diaphoretic. Respiratory examination revealed mild tachypnea with dullness to percussion on right hemithorax, decreased breath sounds in the same area and crackles but no wheezing. Other than the regular tachycardia, the remainder of the physical examination was unremarkable. Initial labs showed leukocytosis of 36.5 K/uL (normal range, 4.5–11k/uL), hemoglobin of 16.7 g/dl (13.5–17.5 g/dl), platelet of 415 K/uL (130–400 k/uL), blood urea nitrogen of 9mg/dl (6–20mg/dl), creatinine of 0.9mg/dl (0.5–0.90 mg/dl), D-Dimer of 492 ng/ml (<250 ng/ml), lactic acid of 1.8 mmol/L (0.5–2.2 mmol/L), BNP (Brain Natriuretic Peptide) of 10 pg/ml (<100 pg/ml). Other tests, including rapid influenza, urine legionella antigen, urine streptococcus pneumoniae antigen, troponin and HIV were negative. A chest X-ray showed consolidation on the right side (Figure 1).\n\nComputerized tomography (CT) angiography of the chest was negative for pulmonary embolism but showed large volume complex parapneumonic effusion on the right side (Figure 2).\n\nThe patient was admitted to the Intensive Care Unit for acute hypoxic respiratory failure and sepsis secondary to right-sided multifocal pneumonia with complex parapneumonic effusion. A chest tube was placed on day 1 with drainage of almost 2.2 L of serosanguinous fluid. Tissue plasminogen activator (10mg every 12 hourly) and deoxyribonuclease therapy (5mg every 12hrly) were given through the chest tube for a total of three days. Pleural fluid was exudative with polymorphonuclear cells. Pleural fluid cultures including mycobacterial cultures, fungal cultures and parasitic exams were negative. Blood culture from day 1 grew G. adiacens in anaerobic bottles sensitive to Ceftriaxone, Levofloxacin, and Vancomycin. Transesophageal echocardiogram (on day 8) did not show any vegetation.\n\nOn day 1 of hospitalization, the patient was started on vancomycin (1250mg every 12 hourly; goal trough of 15–20 mcg/ml), Piperacillin/Tazobactam (3.375 gm every 8 hourly) and azithromycin (500mg daily empirically), which was continued until day 5. On day 6, antibiotics were switched to Ceftriaxone (2 gram IV every 24 hourly), Ampicillin (2 gram IV every 4 hourly), Gentamicin (80mg IV every 8 hourly; goal trough <1 mcg/ml and goal peak 3–4 mcg/ml) and Metronidazole (500mg IV every 8 hourly) for complex parapneumonic effusion with bacteremia secondary to G. adiacens, as per recommendations from the Infectious Disease team, which was continued up to day 21.\n\nThe patient started feeling better with resolution of his fever, cough, chest pain, fatigue and anorexia on day 7. By day 10 his shortness of breath resolved, and he was back to his baseline. His leukocytosis improved from 35.6 k/ul on day 1 to 16.7 k/ul on day 9 and resolved to 9.14 k/ uL on day 17. Follow up CT scan chest on day 15 showed small loculated effusion, which had improved from admission (Figure 3).\n\nThe CT surgery team evaluated the patient and deferred video-assisted thoracoscopic surgery decortication. On day 21, the patient left against the medical advice. The patient completed one more week of Ceftriaxone (2 gram every 24 hourly) and Metronidazole (500mg every 8 hourly) as an outpatient with sustained remission of symptoms on follow-up. A follow-up CT scan of the chest two months later showed a near complete resolution of the residual effusion (Figure 4).\n\n\nDiscussion\n\nA new type of Viridans group Streptococci was discovered in 1961 by Frenkle and Hirsch. This was termed as NVS (Nutritionally Variant Streptococci) and the discovery was based on prolonged incubation period, characteristic growth requirement, variable gram stain findings and satellite promoting phenomenon around cologies of other bacteria . The growth of NVS is not supported by routinely used blood culture media unless they are supplemented with pyridoxal. That is why NVS are usually considered to be very fastidious3. Identification of Granulicatella isolates are performed by either biochemical testing such as API Strep or bioMerieux or molecular confirmation. Identification was delayed in several cases, as isolates were thought to be poorly growing streptococci before consideration of pyridoxal dependence4. Though it is a normal commensal in the oral cavity, the spectrum of infections with Granulicatella species seems to be expanding, and infections are associated with significant morbidity and mortality. The clinical course is more severe than Viridans streptococci. A high index of suspicion and vigilance is needed because of its fastidious nature, slow growth rate, difficulty in isolation on culture media, and multiple antibiotic resistance. Microbial communities that have any kind of impact on tumor progression and microorganisms associated with tumors have been defined as oncobiome. The detailed analysis of microbiome in lung cancer patients documents the presence of G. adiacens, which is not a normal commensal in healthy lungs and further research into this area is warranted5.\n\nThere are reports of G. adiacens as a rare cause of neonatal pneumonia6,7. We could not find any reported cases of pneumonia caused by G. adiacens in immunocompetent adults; however, it has been reported in patients suffering from pulmonary tuberculosis and concurrent HIV infection8. To the best of our knowledge, our case could be the first case of a parapneumonic effusion caused by infection with G. adiacens. Patients at risk of developing Granulicatella species infections are usually immunocompromised in contrast to our patient who was immunocompetent2,9.\n\nBoth G. adiacens (susceptibility rates of 0–63% to ceftriaxone, 81% to amoxicillin, 55–67% to penicillin and 96% to meropenem) and G. elegans (susceptibility rates of 0% to cefuroxime, 100% to penicillin and 33% to ceftriaxone) were reported to have the resistance to β-lactam antibiotics. Resistance was also reported to tetracycline, clindamycin, ciprofloxacin, erythromycin, but not to rifampicin or vancomycin10,11. The infection is usually treated with the antibiotic regimen used for enterococcal endocarditis usually consisting of a combination of ampicillin or penicillin plus gentamicin. Ceftriaxone plus gentamicin or vancomycin monotherapy are reasonable alternatives12.\n\n\nConclusions\n\nGranulicatella adiacens is a fastidious organism that has the potential to infect multiple systems in both immunocompetent and immunocompromised patients.\n\nGiven our case of G. adiacens complex parapneumonic effusion/pneumonia and bacteremia in an otherwise immunocompetent adult and the difficulty to diagnose, a high index of suspicion is required to catch and treat it early on.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for the publication of this case report and any associated images.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nElfessi Z, Liu E, Dukarevich Y, et al.: Sepsis induced bacterial peritonitis caused by Granulicatella adiacens. Am J Emerg Med. 2019; 37(12): 2263.e1–2263.e3. PubMed Abstract \| Publisher Full Text\n\nSenn L, Entenza JM, Greub G, et al.: Bloodstream and endovascular infections due to Abiotrophia defectiva and Granulicatella species. BMC Infect Dis. 2006; 6: 9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPadmaja K, Lakshmi V, Subramanian S, et al.: Infective endocarditis due to Granulicatella adiacens: a case report and review. J Infect Dev Ctries. 2014; 8(4): 548–550. PubMed Abstract \| Publisher Full Text\n\nCargill JS, Scott KS, Gascoyne-Binzi D, et al.: Granulicatella infection: diagnosis and management. J Med Microbiol. 2012; 61(Pt 6): 755–761. PubMed Abstract \| Publisher Full Text\n\nKovaleva OV, Romashin D, Zborovskaya IB, et al.: Human lung microbiome on the way to cancer. J Immunol Res. 2019; 2019: 1394191. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAyub MR, Noor F: Granulicatella adiacens: a rare cause of neonatal pneumonia. J Fatima Jinnah Med Univ. 2018; 12(4). Reference Source\n\nLing Y, Su Q, Jiang S, et al.: Granulicatella adiacens: an unusual cause of early-onset pneumonia in a neonate. Clin Microbiol News. 2016; 38(3): 24–25. Publisher Full Text\n\nMvelase NR, Marajh K, Hattingh O, et al.: An unusual case of thoracic empyema caused by Granulicatella elegans (nutritionally variant streptococci) in a patient with pulmonary tuberculosis and human immunodeficiency virus infection. JMM Case Rep. 2016; 3(5): e005058. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrouqui P, Raoult D: Endocarditis due to rare and fastidious bacteria. Clin Microbiol Rev. 2001; 14(1): 177–207. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTuohy MJ, Procop GW, Washington JA: Antimicrobial susceptibility of Abiotrophia adiacens and Abiotrophia defectiva. Diagn Microbiol Infect Dis. 2000; 38(3): 189–191. PubMed Abstract \| Publisher Full Text\n\nZheng X, Freeman AF, Villafranca J, et al.: Antimicrobial susceptibilities of invasive pediatric Abiotrophia and Granulicatella isolates. J Clin Microbiol. 2004; 42(9): 4323–4326. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaddour LM, Wilson WR, Bayer AS, et al.: Infective endocarditis in adults: diagnosis, antimicrobial therapy, and management of complications: a scientific statement for healthcare professionals from the American Heart Association. Circulation. 2015; 132(15): 1435–1486. PubMed Abstract \| Publisher Full Text" }	[ { "id": "153445", "date": "10 Nov 2022", "name": "Motoi Ugajin", "expertise": [ "Reviewer Expertise Respiratory infections", "lung malignancies", "and interstitial pneumonia." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 intend to show G.adiacens can cause complicated parapneumonic effusion with bacteremia through this case report. However, I feel there are several flaws on the current report. Now I will mention my concerns for this report:\n1) Please show the detailed information about right pleural effusion. The definition of complicated parapneumonic effusion requires lactate dehydrogenase level and glucose level.\n2) In this case, blood culture showed the growth of G.adiacens, while pleural effusion could not reveal the growth of this bacteria. In the discussion section, the authors mention routinely used blood culture media does not support the growth of G.adiacens. Nevertheless, the case could obtain the growth of G.adiacens on blood culture. The authors should describe the reasons why the authors could obtain the growth of this bacteria on blood cultures and why the authors could not reveal the growth of this bacteria on pleural effusion.\n3) In the discussion section, the authors mention the clinical course of infection due to G.adiacens is more severe than Viridans streptococcus. Please show the concrete information which can support your opinion.\n4) The authors mention this patient is immunocompetent. However, this patient is reported to be a chronic alcoholic. Some previous literature has shown alcoholic patients are susceptible to respiratory infections1,2. Indeed, the definition of immunocompromised is unclear, but I think it is difficult to categorize this patient as immunocompetent.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] }, { "id": "160513", "date": "23 Jan 2023", "name": "Werner Albrich", "expertise": [ "Reviewer Expertise Infectious Diseases", "respiratory tract infections", "antibiotic therapy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this case report the authors describe a patient with bacteremia and a pneumonia with a parapneumonic effusion caused by Granulicatella adiacens.\nMajor comments:\nThe diagnosis (in title, abstract and main text) should include pneumonia. There was a description of consolidation supporting the diagnosis of pneumonia on chest X-ray. Was this also seen on the chest CT scan, prior to or after drainage of the pleural effusion? If so, this should be added. The text described it as multifocal pneumonia. Common pneumonia classifications use rather multilobar (if this was the case). Which lobe was affected as this might give a clue towards aspiration? In addition, the case report does not contain information which allows to confirm that it is a complex parapneumonic effusion versus a pleural empyema. Was there septation or inhomogeneity on chest CT? Were pH, LDH, protein/albumin measured? Were pleural cultures obtained prior to or after start of antibiotics? Given the reported data, the patient was not septic according to the Sepsis-3 definition, in fact, he was hypertensive.\n\nThe patient is a chronic alcoholic and smoker and may differ therefore from a healthy person. Therefore, it seems more accurate to state this rather than to call him an immunocompetent host (in title, abstract, case presentation and discussion). Was there any clue for aspiration pneumonia?\n\nWas there continuous bacteremia, i.e. follow-up blood cultures?\n\nWas there susceptibility testing for penicillin/ampicillin and gentamicin or other aminoglycosides? I cannot follow the rational to combine ampicillin with penicillin (as stated in the abstract) and would reformulate “ampicillin or penicillin plus gentamicin”. The antibiotic regimen is unusual in several aspects:\nWas there a loading dose for vancomycin? The piperacillin dosage is rather low if there was normal renal function. Given concerns for acute renal failure with the combination of vancomycin with piperacillin/tazobactam, this combination is falling out of favour and is very broad for a patient not at risk for resistant pathogens and not in septic shock. The targeted quadruple therapy is not explained by the susceptibility testing. Why was ceftriaxone combined with ampicillin? And if ceftriaxone is used why was gentamicin added and why 3x/d rather than the less nephrotoxic once daily dose? Also, the addition of metronidazole can be viewed critically if there is no evidence of empyema or aspiration. If there was no empyema (and no sign of endocarditis), the treatment duration of 4 weeks is much longer than currently recommended in guidelines for pneumonia. In view of the threat of antibiotic resistance and to support antibiotic stewardship, this therapy should be critically discussed.\n\nMinor comments:\nAbstract:\nGranulicatella adiacens is not formally any more a streptococcus but belongs a separate genus (https://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&search_value=962037#null ).\nIntroduction:\nThe pathogen should be more properly described as “the fastidious Granulicatella genus previously considered as nutritionally variant group streptococci”.\nCase presentation:\nI am not sure if previous phenytoin use is important. Should use the name of the drug rather than the trade name. If known, should add the actual number of breaths per second to characterize the tachypnea. Was sputum cultured? Was there testing for SARS-CoV-2 or was this prior to the pandemic? Purohit et al1 also describe a case of empyema with Granulicatella adiacens, so this is likely not the first case.\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/9-977
https://f1000research.com/articles/9-975/v1	12 Aug 20	{ "type": "Research Article", "title": "Cytotoxic effects of manganese oxide nanoparticles in combination with microbial components on intestinal epithelial cells", "authors": [ "Jorrell Fredericks", "Sujata Senapati", "Michael J. Wannemuehler", "Jorrell Fredericks", "Sujata Senapati" ], "abstract": "Background: Manganese oxide has been shown to cause toxicity and is associated with occupational-related disease (e.g., welders). With the goal to improve several biomedical areas, manganese oxide nanoparticles (MnO NP) are being considered for use in drug delivery and magnetic resonance imaging (MRI) to obtain high resolution anatomical images of tumors and gastrointestinal (GI) inflammation. Regardless of whether it is intentional or unintentional ingestion, the GI tract has been shown to be the primary route of entry for metal nanoparticles including MnO NP. However, studies assessing toxicity of MnO NP for intestinal epithelial cells (IECs) are virtually nonexistent. Methods: Given the proximity to the GI lumen, assessing the effects of nanoparticles on IECs in the presence of bacterial components presents a more holistic model of exposure. Therefore, we examined the effects of MnO NP alone and MnO NP in combination with Escherichia coli LF82 bacterial lysate on selected functions of MODE-K cells, a murine intestinal epithelial cell line. Data were analyzed using one-way ANOVA. Differences with p < 0.05 were considered significant. Results: Results showed MnO NP plus E. coli LF82 lysate added to MODE-K cells severely inhibited monolayer scratch wound healing, enhanced the secretion of interleukin 6 (IL-6), and induced mitochondrial dysfunction. Conclusions: Overall, our findings show that toxicity of MnO NP deleteriously affected MODE-K cells and demonstrated the necessity to integrate other environmental factors, such as microbial components and/or inflammatory cytokines, into studies assessing effects of nanoparticles on mucosal epithelia.", "keywords": [ "MnO nanoparticles", "epithelial cells", "gastrointestinal", "inflammatory bowel disease", "mitochondrial function" ], "content": "Abbreviations\n\nManganese oxide nanoparticles (MnO NP), intestinal epithelial cells (IECs), gastrointestinal (GI), inflammatory bowel disease (IBD), magnetic resonance imaging (MRI), tight junction (TJ), oxygen consumption rates (OCR), interleukin 6 (IL-6), reactive oxygen species (ROS)\n\n\nIntroduction\n\nThe gut mucosa continuously encounters a barrage of biological challenges including those that are derived from the host, microbial and foreign/food antigens, environmental toxicants, and/or biological toxins. The intestinal epithelial barrier is also the first line of physical defense in the intestine. Intestinal epithelial cells (IECs) sense foreign and endogenous danger signals and plays an important role in the maintenance of mucosal immune homeostasis by regulating inflammation and restricitng the uptake of phlogistic components from the lumen1–4. Consequently, IECs are at the forefront of the physiological barrier separating the host from the inflammatory components present in the intestinal lumen. As the intestinal barrier is constantly faced with steady insult from microbial and other environmental influences, there are many challenges that affect the maintenance of mucosal homeostasis. For example, the resident microbiota, that inhabit the gut, plays a critical role in several IEC processes, including the rate of intestinal epithelial cell turnover, promotion of epithelial restitution after injury, and reorganization of tight junction (TJ) proteins which all contribute to a healthy epithelial barrier3. However, there are also deleterious outcomes that can be induced by microbial components and excreted proteins. Microbial enterotoxin have been shown to interact and interfere with TJ integrity which negatively affects the epithelial integrity by increasing the exposure of the host to microbial-derived inflammatory components4.\n\nAs chronic inflammation is one of the main pathological features of inflammatory bowel disease (IBD), the intestinal epithelial barrier integrity plays a major role in maintaining a balance between host–microbial interactions that can affect susceptibility to and the pathogenesis of IBD5. Disruption of the intestinal barrier, along with increased levels of proinflammatory cytokines, has been shown to lead to or be present at the onset of intestinal inflammation and disorders including IBD6–8. The presence of proinflammatory cytokines in a healthy intestine are beneficial (i.e., cytoprotective) based on their role in regulating a variety of biological processes including IEC activation, replication, differentiation, as well as the regulation of innate and adaptive immunity9. However, the imbalance between pro-inflammatory and anti-inflammatory cytokines impedes the resolution of inflammation and adversely affects the maintenance of the intestinal homeostasis leading to the perpetuation of disease and tissue destruction10.\n\nNanotechnology is a rapidly growing field that has been shown to provide many advantages in various areas of medicine. With the goal to improve several technological areas, such as drug delivery and magnetic resonance imaging, the usage of nanoparticles, specifically metal nanoparticles including manganese oxide nanoparticles (MnO NP), have played in important role in advancing these fields11–16. Despite the potential benefits, there are also concerns about the induction of adverse health effects17. With the growing concerns regarding the unintentional environmental effects of nanoparticles on the gastrointestinal (GI) tract, there needs to be further evaluation of the effects that MnO NP may have on biological systems18. For example, metal-based NPs could contribute to adverse health conditions resulting from alterations of gene expression, dysregulation of physiological processes due to improper intracellular trafficking, and accumulation of NPs at toxic concentrations19. More complete understanding of the basis of metal-based NPs toxicity is essential to elucidate the occupational, environmental, and health risk-assessments. Regardless of whether it is intentional or unintentional ingestion, the GI tract has been shown to be the primary route of entry for metal nanoparticles including MnO NP. Individuals can be exposed to MnO NPs as a result of their occupation (e.g. welding) or medical treatment (e.g. magnetic resonance imaging (MRI))20–22. Given the proximity to the GI lumen, assessing the effects of nanoparticles on IECs in the presence of bacterial components presents a more holistic model of exposure when assessing the effects of MnO NP on gut IECs as the gut microbiota influence cellular physiology, metabolism, and immune function23. However, published studies that take into consideration the interaction between host IECs, nanoparticles, and microbiota are extremely limited. Therefore, the objective of this study is to investigate the cytotoxicity of environmentally related MnO NP on IECs. To mimic the in vivo environment, these studies were designed to examine the interactions of IECs with MnO NP in conjunction with a bacterial lysate derived from E. coli LF82. Results indicate there are synergistic interactions between MnO NPs and bacterial components that adversely affect IEC function.\n\n\nMethods\n\nManganese oxide nanoparticles (MnO NP (US Research Nanomaterials, Inc., Houston, TX, USA)) used in all experimental procedures were kindly provided by Dr. A. Kanthasamy (Department of Biomedical Sciences, Iowa State University, Ames, IA). Based on transmission electron microscopy the size of MnO NP were 16.8 ± 2.6 nm in diameter.\n\nThe MODE-K intestinal epithelial cell line generated from the small intestine of a C3H mouse was used in all experimental procedures described below (kind gift from Dr. C. Elson, Division of Gastroenterology and Hepatology, School of Medicine, University of Alabama at Birmingham, AL). In this study, MODE-K cells were grown in high glucose (4.5 g/L) DMEM medium containing 10% FBS, 2 mM L-glutamine, 50 units of penicillin, and 50 µg/mL streptomycin. In general, cells were inoculated into a tissue culture plate (6–96 wells) depending on the experimental requirements and were cultured overnight in a humidified atmosphere of 5% CO2 at 37°C. The length of each experiment and MODE-K cell density for each experiment has been provided in the methods section for each experimental procedure. For the majority of experiments, there were four treatment groups: control, 1 µg/mL E. coli LF82 lysate, 10 µg/mL MnO NP, and 10 µg/mL MnO NP plus 1 µg/mL E. coli LF82 lysate. At 24 hours before initiating experimental treatments, the culture medium was switched from high-glucose medium to low-glucose DMEM medium. Dose titration experiments were performed to identify the minimal dose of E. coli LF82 lysate and the MnO NPs that did not affect cell viability after 24 hours, and these doses were chosen to assess the interactions between the MnO NPs and E. coli LF82 lysate.\n\nAn adherent invasive E. coli (AIEC) strain LF82 that has been associated with ileal Crohn’s disease was cultivated in BHI broth at 37°C in an aerobic environment. Bacteria were harvested after overnight replication, washed with PBS, frozen, lyophilized, and stored at -20°C until used. The lyophilized bacterial cells were weighed and resuspended in sterile PBS (2 mg/mL) prior to being subjected to two freeze-thaw cycles, followed by sonication at 50 amps, 75 amps, and 100 amps for 30 seconds at each amperage. This sonication cycle was repeated three times. The lysates were sterilized by ultraviolet irradiation and sterility confirmed bacteriologically. With minor modifications, this method was used as previously described24.\n\nCell viability was measured using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation (MTS assay kit from Promega Corporation, Madison, WI). MODE-K cells were inoculated into 24-well plates with 120,000 cells/mL per well one day prior to treatment. The next day, MODE-K cell monolayers were washed twice with low glucose DMEM before addition of the respective stimulants diluted in low glucose DMEM medium and cultured for an additional 48 hours. Following treatment, 20 µL of MTS solution reagent mix was added to each well and incubated at 37°C for 30 minutes. At the end of incubation, readings were taken at a wavelength of 490 nm and another reference reading for each well was taken at 670 nm to eliminate background. This method was previously described and adopted to fit our experimental procedure25. Data shown is represented as the mean ± SEM collected from two independent experiments.\n\nPrior to assessing cell viability, supernatants were collected from MODE-K cell cultures at 24 and 48 hours after addition of the MnO NP and/or E. coli LF82 lysate and analyzed for the presence of cytokines. A multiplex bead-based assay was used to measure the presence of IL-6, TNFα, and IFNγ. The assay was performed using Bio-Plex Pro COOH magnetic beads (Luminex Corp., Austin TX) conjugated with the following monoclonal antibodies (Fisher Scientific Co. LLC) to capture IL-6 (Cat # 14-7061-85; clone MP5-20F3), TNFα (Cat # 14-7313-81, clone AN-18), or IFNγ (Cat. # 14-7325-85; clone 1F3F3D4). Biotinylated monoclonal antibodies (Fisher Scientific Co. LLC) were used for detection of the bound (i.e., captured) IL-6 (Cat. # 13-7062-85; clone MP5-32C11), TNFα (Cat # 13-7326-85; clone MP6-Xt3), and IFNγ (Cat. # 13-7312-85; clone R4-6A2). Mean fluorescence intensity was measured using Strep-Avidin conjugated phycoerythrin (PE) (Fisher Scientific Company, LLC) and a Bio-Plex System 200 (BioRad Life Science, Hercules, CA). Standard curves were generated using commercially available cytokine standards (Peprotech, Inc., Cranbury, NJ) and Bio-Plex Manager Software Version 6.0 (Bio-Rad Laboratories, Inc., Carlsbad, CA, USA). Results are representative of two independent experiments containing six replicates per treatment.\n\nFor one-dimensional migration assays, 300,000 MODE-K cells per well were suspended in DMEM (low glucose, 1.0 g/L) inoculated into 12 well plates and incubated at 37 °C in 5 % CO2 overnight. The next day a (+) shaped scratch (i.e., monolayer wound) was created in each well using a P-200 pipette tip, creating a cell free gap that, in time, would be covered by replicating/migrating MODE-K cells. Treatments were then added and cells were then incubated for 24 hours. Images were taken at 0 and 24 hours post-wounding. The four treatment groups were: medium alone (i.e., control), 1 µg/mL E. coli LF82 lysate, 10 µg/mL MnO NP, and 10 µg/mL MnO NP plus 1 µg/mL E. coli LF82 lysate. After 24 hours, experiments were terminated. Images were taken using a 5x lens on an inverted microscope Leica DMi1 (Leica Microsystems, IL, US) using the software Leica Application Suite 4.5.0 (Leica Microsystems, IL, US) from 10 representative wounded areas per treatment. Images and gap spacing were then measured and analyzed with an automation and image analysis software Metamorph 5.0.7 (Molecular Devices LLC, CA, US). (ImageJ is a freely available alternative that can be used for the same purposes as Metamorph.) Gap closure was quantified by determining the mean distance of closure (i.e., healing) of the gap. This was done by measuring the area of the cell-free space separating the two edges of the cellular monolayer at 24 hours after addition of the treatments, and then subtracting this value from starting value that was measured at the 0-hour time point. This method was previously described and adopted to fit our experimental procedure26. Results are a representation of two independent experiments containing six replicates per treatment.\n\nMitochondrial superoxide generation was measured using MitoSOXTM Red Mitochondrial Superoxide Indicator (Thermo Scientific). The four treatment groups were: medium alone, 1 µg/mL E. coli LF82 lysate, 10 µg/mL MnO NP, and the 10 µg/mL MnO NP plus 1 µg/mL E. coli LF82 lysate. MODE-K cells were plated in 96-well plates with 8,000 cells/well one day prior to treatment and allowed to attach overnight. The next day, cells were washed twice with low glucose DMEM medium before addition of the indicated treatments suspended in low glucose DMEM medium and the cells were incubated for an additional 24 hours. Following treatment, MitoSox reagent (Thermo Scientific) was added to each plate well at a 1:1000 concentration. Relative fluorescence intensity was then acquired using a microplate reader per manufacturers instructions. Data is representative of two independent experiments containing six wells per treatment per experiment and is presented as the mean ± SEM.\n\nMitochondrial respiration was measured using a Seahorse XF24e Extracellular Flux analyzer (Seahorse Bioscience, North Billerica, MA). The Seahorse XF24e Extracellular Flux analyzer is a high-throughput instrument that yields real-time measurements of cellular respiration rates. In our studies, we were interested in assessing the effects of four treatments on mitochondrial respiration and their effects on oxygen consumption rates (OCR). MODE-K cells were seeded at a density of 300,000/mL into a T-25 culture flask and incubated overnight in 5% CO2 at 37.8ºC. The next day, cells were treated with respective treatments for 24 hours. Stimulated cells were recovered from the plates by scraping and added to the Seahorse plates using CellTac at 60,000 cells per well and incubated for 1 hour to allow the cells to adhere. At this time, the mito-stressor reagents oligomycin (1 µM), FCCP (2 µM) and rotenone/antimycin A (0.5 µM) were loaded into the corresponding injection ports of the Seahorse FluxPak cartridge and placed in the Seahorse analyzer to be equilibrated. After equilibration, the plate containing treated cells was then placed in the Seahorse analyzer covered with the Flux Pak cartridge. The analyzer was then programmed to measure the basal OCR readouts at five specified time intervals before progressing to inject the mito-stressors every three cycles of measuring OCR. Measuring mitochondrial respiration with the Seahorse analyzer allowed the measurement of the effect of each treatment at the three critical stages of cellular respiration. Data shown is the mean ± SEM and are representative of five independent wells.\n\nAll statistical data analyses were performed using Prism 4.0 (GraphPad Software, San Diego, CA). Data were analyzed using one-way ANOVA with the Tukey post-hoc test for comparing all treatment groups with that of the control. Multiple comparisons test was also performed to analyze differences between treatment groups. Differences with p < 0.05 were considered significant. All data is representative of two independent experiments containing six wells per treatment.\n\n\nResults\n\nThe induction of reactive oxygen species (ROS) may negatively impact the crucial barrier function of the intestinal epithelium. Reactive oxygen species are increased in the inflamed mucosa of IBD patients and may contribute to loss of intestinal barrier function27. Furthermore, during inflammation, oxidative stress can lead to the induction of cell injury and apoptosis of intestinal epithelial cells28.\n\nTo assess the induction of mitochondrial ROS in the presence of MnO NPs, the MitoSOX assay was used. After 24 hours of incubation with the indicated treatments, measurements were taken and data was normalized as percent control (Figure 1). Although there was no significant difference, there was approximately a 15% or 25% increase in mitochondrial superoxide generation in treatment groups receiving MnO NP or MnO NP + bacterial lysate, respectively (Figure 1). While there was a slight additive effect, this data suggests that mitochondrial superoxide generation by the MODE-K cells was primarily driven by the presence of MnO NP as opposed to the E. coli LF82 lysate.\n\nData was normalized to percent control. MODE-K cells were incubated in the presence of 1 µg/mL E. coli LF82 lysate (LF82), 10 µg/mL MnO nanoparticles (NP), or a combination of both. Data is representative of two independent experiments containing six wells per treatment per experiment and is presented as the mean ± SEM.\n\nDamage and impairment of the intestinal epithelial barrier may result in an increased permeability and absorption of toxic and phlogistic factors from the intestinal lumen leading to inflammation and disequilibrium of epithelial homeostasis29. This inflammatory response can lead to the induction of cellular injury and apoptosis of intestinal epithelial cells28,29. To assess the secretion of inflammatory cytokines from the MODE-K cells, culture supernatants were harvested 24 hours after the addition of the treatments. IL-6 was the only cytokine detected (i.e., no TNFα or IFNγ) and this was associated with exposure to the E. coli LF82 lysate as opposed to the MnO NP alone. However, a significant in IL-6 secretion was detected when the MODE-K cells were treated with both the E. coli LF82 lysate + MnO NP (p < 0.05) (Figure 2A). Similar results were also obtained in culture supernatants collected after 48 hours of incubation (Figure 2B); however, there was a threefold increase in the secretion of IL-6 at 48 hours when the MODE-K cells were exposed to both stimulants.\n\n(A) Culture supernatants were harvested at 24 hours post-treatment. (B) Culture supernatants were harvested at 48 hours post-treatment. Results are representative of two independent experiments containing six replicates per treatment. Different letter denotes difference in significant (p < 0.05) change. The error bars represent the SEM.\n\nWound healing is a crucial function of the intestinal epithelium as it can be injured by toxic luminal substances, inflammation, interactions with microbes, oxidative stress, and pharmaceuticals27,30. Upon injury, the intestinal epithelium undergoes a wound healing process which is dependent on the balance of three cellular events; restitution, proliferation, and differentiation of immature epithelial cells adjacent to the wounded area31. To assess the effects of MnO NP on the wound healing process (i.e., replication and migration), a scratch wound assay using MODE-K cell monolayers was performed. Using a one-way ANOVA, a significant effect was observed between treatment groups and controls (p < 0.05) (Figures 3A and 3B).\n\nMeasurements of the gaps created by the scratch were taken at 0 and 24 hours post-treatment. (A) Photographs of representative monolayers taken 24 hours post-treatment. (B) Histogram depicting the distance traveled (µm) by MODE-K cells to close the gap between edges of the wound. Results are a representation of two independent experiments containing six replicates per treatment. Different letter denotes difference in significant (p < 0.05) change. Error bars represent the SEM.\n\nPost-hoc analysis indicated a significantly (p < 0.05) slower wound healing response when the control cells (medium alone) was compared to MODE-K cells treated with either 10 µg/mL MnO NP alone or 10 µg/mL MnO NP + 1 µg/mL E. coli LF82 lysate. In contrast, the addition of the E. coli LF82 lysate alone to the cultures had no effect on the replication and migration of the MODE-K cells. However, analysis revealed a significant (p < 0.05) decrease in wound healing when cells were treated with 10 µg/mL MnO NP + 1 µg/mL E. coli LF82 lysate compared to the 10 µg/mL MnO NP alone treatment group suggesting a synergistic effect on wound healing.\n\nMitochondrial function plays a critical role in maintaining intestinal stemness and homeostasis32. As mitochondrial dysfunction is associated with a loss of proliferative capacity by intestinal epithelial cells, the effects on crucial factors of mitochondrial respiration were measured using a mitostress test. Utilizing the Seahorse XFe24 Analyzer, changes in basal respiration, maximal respiration, proton leak, ATP production, and spare respiration capacity were measured (Figure 4).\n\nAfter incubation in the presence of 1 µg/mL E. coli lysate, 10 µg/mL MnO NP or both, metabolic responses of MODE-K cells was measured as described in the Methods. Mitochondrial stress test (MST) was used to assess the oxygen consumption rate (OCR) including the basal respiration (Figure 5), maximal respiratory capacity (Figure 6), proton leak (Figure 7), ATP production (Figure 8) and spare respiratory capacity (Figure 9). Data shown is a single experimental replicate that is representative of two independent experiments.\n\nAfter incubation in the presence of 1 µg/mL E. coli LF82 lysate, 10 µg/mL MnO NP or both, metabolic responses of MODE-K cells was measured as described in the Methods. Data shown is the mean ± SEM of five independent wells. Significant (p < 0.05) difference is denoted by difference in letter.\n\nAfter incubation in the presence of 1 µg/mL E. coli LF82 lysate, 10 µg/mL MnO NP or both, metabolic responses of MODE-K cells was measured as described in the Methods. Data shown is the mean ± SEM and are representative of five independent wells. Significant (p < 0.05) difference is denoted by difference in letter.\n\nAfter incubation in the presence of 1 µg/mL E. coli LF82 lysate, 10 µg/mL MnO NP or both, metabolic responses of MODE-K cells was measured as described in the Methods. Data shown is the mean ± SEM and are representative of five independent wells. Significant (p < 0.05) difference is denoted by difference in letter.\n\nAfter incubation in the presence of 1 µg/mL E. coli LF82 lysate, 10 µg/mL MnO NP or both, metabolic responses of MODE-K cells was measured as described in the Methods. Data shown is the mean ± SEM and are representative of five independent wells. Significant (p < 0.05) difference is denoted by difference in letter.\n\nAfter incubation in the presence of 1 µg/mL E. coli LF82 lysate, 10 µg/mL MnO NP or both, metabolic responses of MODE-K cells was measured as described in the Methods. Spare capacity of MODE-K cells were measured at 24 hours post-treatment initiation. Data shown is a representation of five independent wells. Significant (p < 0.05) difference is denoted by difference in letter.\n\nBasal respiration is a measurement that shows the energetic demand of the cell under baseline conditions. Analysis showed a significant (p < 0.05) elevation of basal respiration in both treatments groups containing MnO NP when compared to medium and E. coli LF82 lysate alone, with the highest increase induced by the MnO NP alone (Figure 5).\n\nMaximal mitochondrial respiration, which is a measurement of the maximum rate of respiration that cells can achieve when operating at maximum capacity was alsao measured. Significantly (p < 0.05) lower levels of maximal respiration were seen in cells treated with the MnO NP alone and an even more severe affect was observed in the MnO NP + E. coli LF82 lysate treated group (Figure 6). While not significant, MODE-K cells treated with both MnO NP + E. coli LF82 lysate, experienced a greater decrease in maximal respiration indicating that the presence of bacterial lysate further impacted mitochondrial respiration.\n\nAnother parameter measured in the mitostress test (MST) is mitochondrial proton leak. Mitochondrial proton leak can have a major impact on mitochondrial oxidative phosphorylation coupling efficiency and production of reactive oxygen species. The presence of MnO NP caused a significant increase (p < 0.05) in mitochondrial proton leak either alone or in combination with the E. coli LF82 lysate (Figure 7).\n\nATP production was also measured during the MST and it was observed that MnO NP induced higher oxygen consumption rates, as evidenced by the significant (p < 0.05) increase in ATP production when compared to control and MODE-K cells treated with the E. coli LF82 lysate alone. In addition, the presence of the bacterial lysate appeared to attenuate the metabolic activity induced MnO NP alone (Figure 8). Any treatment group containing MnO NP experienced an increase in ATP production suggesting this to be a MnO NP-mediated effect.\n\nThe final parameter measured in the MST was spare respiratory capacity, which measures the capability of the cell to respond to an energetic demand, which can be an indicator of the cell’s fitness or flexibility. When compared to control MODE-K cells, any group treated with MnO NP had a significant (p < 0.05) decrease in the spare respiratory capacity suggesting that a decrease in respiratory spare capacity was mediated by the presence of the MnO NPs independent of the presence of E. coli LF82 lysate (Figure 9).\n\nMitochondrial dysfunction is associated with a loss of proliferative capacity in intestinal epithelial cells32. As the MTS assay takes into account three parameters (cytotoxic, cytostatic, and antiproliferative effects), any decrease in the presence of viable cells may be a result from either one or a combination of these three parameters and would provide crucial insight about cell viability and health. After 48 hours of incubation, a significant decrease was observed in the MnO + E. coli treated MODE-K cells (p < 0.05) (Figure 10) which is consistent with the wound healing data shown in Figure 3. There was no significant impact measured by the MTS when the cells were treated with either the MnO NP or E. coli LF82 lysate alone.\n\nThe viability of MODE-K cells was measured 48 hours after the addition of 1 µg/mL E. coli LF82 lysate, 10 µg/mL MnO NP or both. Data shown is represented as the mean ± SEM collected from two independent experiments. In each experiment, six replicate wells were measured per treatment with significant (p < 0.05) difference denoted by difference in letter.\n\n\nDiscussion\n\nThe intestinal epithelial barrier, which consists of several elements, including intestinal epithelial cells and tight junctions, prevents microorganisms, undesirable solutes, toxins, and luminal antigens from entering the body33–37. It is suggested that disruption of the intestinal barrier function and repeated intestinal epithelial damage are key features of IBD, as well as other intestinal disorders29,38. Furthermore, it is known that environmental irratants (e.g., pollutants) negatively impact GI IECs. Environmental exposure to MnO NPs can occur as a result of occupational exposure in manganese ore processing, the metallurgy industry, metalworking, welding, and various potential biomedical applications20–22,39. MnO NP have been shown to cause impairment of dopaminergic neurons and induce inflammation in the brain, increase in oxidative stress and apoptosis in alveolar epithelial cells, and increase oxidative stress and DNA damage in mouse embryonic stem cells, human breast cancer epithelial cells, and human fibrosarcoma epithelial cells39–42. However, few studies have evaluated at the effects of MnO NP toxicity on intestinal epithelial cells, even though the primary route of exposure would be oral or ingestion. Therefore, the goal of this study was to investigate the cytotoxicity of environmentally related MnO NP on IECs. As most studies evaluate the biological effects of MnO NP alone, this approach may underestimate their in vivo toxicity in the GI tract given the potential interactions with the resident microbiota. In this study, new insights regarding the toxicity of MnO NP for IECs was evaluated in combination with bacterial components (e.g., E. coli LF82 lysate). The presence of the bacterial products seem to have both negative and cytoprotective effects on the MODE-K cells. By themselves, MnO NP negatively affected MODE-K epithelial cells, but in the presence of the bacterial lysate, synergistic effects were observed in several biological functions including secretion of IL-6, wound healing, and mitochondrial processes.\n\nIn this study, IL-6 secretion was synergistically elevated in the culture supernatant of MODE-K cells stimulated with MnO NP + E. coli LF82 lysate. IL-6 is a pleiotropic, pro-inflammatory cytokine which plays an important role in promoting inflammatory response in the gut and in the systemic circulation10,38. Elevated IL-6 serum levels have been detected in acute and chronic inflammation GI disease. Moreover, elevated IL-6 production has been associated with Crohn’s disease (CD) and ulcerative colitis (UC)10. As elevated levels of localized IL-6 in the intestine may be a characteristic feature of active IBD, IECs are one of the major cell types responsible for the production of IL-643. Modulation of IL-6 levels in the GI tract can undermine the integrity of the intestinal epithelial barrier by increasing intestinal barrier permeability44,45.\n\nBecause manganese ions were detected in culture supernatants (1853 – 2585 ppb), it is possible that excessive Mn++ ions in the culture medium had an impact mitochondrial functions but published data indicates that cytotoxicity occurs at mM concentrations46. Mn++ ions have an essential role in survival and death mechanisms of cells from all organisms because Mn++ ions regulate Mn-containing enzymes such as manganese superoxide dismutase and regulate expression and activity of caspases. Accumulation of Mn++ ions to excessive levels (≥ mM concentrations) has been shown to cause cellular dysfunction and cell death46. Data suggest the presence of MnO NP led to an accumulation and excess level of Mn++ in IECs that disrupted the balance of the redox interaction and caused mitochondrial dysfunction. This would in turn lead to deleterious effects in wound healing response that was observed in this study. Control of intestinal epithelial stemness is crucial for tissue homeostasis and the mitochondrial function plays a critical role in maintaining intestinal stemness32. Dysfunction of the mitochondria was accompanied by impairment in ATP production which resulted in reduced proliferation of the intestinal epithelium and loss of stemness32. The results of this study demonstrated that combination of MnO NP and E. coli LF82 lysate caused impairment of mitochondrial respiration of MODE-K cells. These results suggest that the presence of MnO NP in the gut could affect mitochondrial energy capacity and consequently mucosal homeostasis resulting in impairment of barrier function and development of leaky gut syndrome.\n\nMitochondria play an important role during the interaction of gut microbiota with host cells and mitochondrial activity may be an important factor that modulates microbiota diversity and quality47. A study performed using colonic biopsy samples showed that induced mitochondrial dysfunction affected epithelial barrier function, which allowed transepithelial flux of bacteria across the intestinal epithelium48. An extraordinary amount of energy is required by IEC during the inflammatory and immune responses49. For example, the first contact of the gut microbiota with the intestinal epithelium stimulates Toll-like receptors in IECs which results in the recruitment of B cells and plasma blasts that subsequently secrete IgA to limit the over-colonization of gut microbiota50. Based on the described in vitro studies, it is predicted that environmental exposure to MnO NP will dysregulate the mitochondrial function leading to a loss in epithelial barrier integrity and the infiltration of gut microbes initiating an inflammatory response50.\n\nThe GI tract has been shown to be the primary route of ingestion and excretion for metal nanoparticles. Results of this study suggest that the addition of bacterial components to IEC cultures in combination with MnO NP might be predictive of the cellular response seen in vivo as this more appropriately represents the context of exposure to environmental irratants and toxicants in the GI tract. Currently, the toxicity of nanoparticles is evaluated with various approaches that include both in vitro and in vivo methods of assessment, but rarely do they take into consideration the presence of the microbial community that exist in the body. Assessing the effects of nanoparticles on intestinal epithelial cells in the presence of bacterial components presents a more holistic, and representative model of exposure when assessing the effects of nanoparticles on gut epithelial cells as the gut microbiota can influence cellular physiology, metabolism, and immune function23. The results presented herein indicate that it is important to integrate the contributions of multiple stimuli on IEC function and ultimately intestinal barrier integrity in order to obtain a more representative impact of metal-based nanoparticles on the health and homeostasis of the GI tract.\n\n\nData availability\n\nOpen Science Framework: Cytotoxic effects of manganese oxide nanoparticles in combination with microbial components on intestinal epithelial cells, https://doi.org/10.17605/OSF.IO/2RKEA51\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors thank Mary Jane Long and Meghan Wymore-Brand for technical assistance and Dr. Anumantha Kanthasamy for the kind gift of manganese oxide nanoparticles.\n\n\nReferences\n\nPastorelli L, De Salvo C, Mercado JR, et al.: Central role of the gut epithelial barrier in the pathogenesis of chronic intestinal inflammation: lessons learned from animal models and human genetics. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVongsa RA, Zimmerman NP, Dwinell MB: CCR6 regulation of the actin cytoskeleton orchestrates human beta defensin-2- and CCL20-mediated restitution of colonic epithelial cells. J Biol Chem. 2009; 284(15): 10034–45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIizuka M, Konno S: Wound healing of intestinal epithelial cells. World J Gastroenterol. 2011; 17(17): 2161–71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerger E, Rath E, Yuan D, et al.: Mitochondrial function controls intestinal epithelial stemness and proliferation. Nat Commun. 2016; 7: 13171. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWatson AJ, Chu S, Sieck L, et al.: Epithelial barrier function in vivo is sustained despite gaps in epithelial layers. Gastroenterology. 2005; 129(3): 902–12. PubMed Abstract \| Publisher Full Text\n\nLaukoetter MG, Bruewer M, Nusrat A: Regulation of the intestinal epithelial barrier by the apical junctional complex. Curr Opin Gastroenterol. 2006; 22(2): 85–9. PubMed Abstract \| Publisher Full Text\n\nBrandt EB, Strait RT, Hershko D, et al.: Mast cells are required for experimental oral allergen-induced diarrhea. J Clin Invest. 2003; 112(11): 1666–77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchulzke JD, Bentzel CJ, Schulzke I, et al.: Epithelial tight junction structure in the jejunum of children with acute and treated celiac sprue. Pediatr Res. 1998; 43(4 Pt 1): 435–41. PubMed Abstract \| Publisher Full Text\n\nD'incà R, Di leo V, Corrao G, et al.: Intestinal permeability test as a predictor of clinical course in Crohn's disease. Am J Gastroenterol. 1999; 94(10): 2956–60. PubMed Abstract \| Publisher Full Text\n\nJump RL, Levine AD: Mechanisms of natural tolerance in the intestine: implications for inflammatory bowel disease. Inflamm Bowel Dis. 2004; 10(4): 462–78. PubMed Abstract \| Publisher Full Text\n\nFrick R, Müller-edenborn B, Schlicker A, et al.: Comparison of manganese oxide nanoparticles and manganese sulfate with regard to oxidative stress, uptake and apoptosis in alveolar epithelial cells. Toxicol Lett. 2011; 205(2): 163–72. PubMed Abstract \| Publisher Full Text\n\nLi T, Shi T, Li X, et al.: Effects of Nano-MnO2 on dopaminergic neurons and the spatial learning capability of rats. Int J Environ Res Public Health. 2014; 11(8): 7918–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMccarrick S, Cappellini F, Kessler A, et al.: ToxTracker Reporter Cell Lines as a Tool for Mechanism-Based (geno)Toxicity Screening of Nanoparticles-Metals, Oxides and Quantum Dots. Nanomaterials (Basel). 2020; 10(1): 110. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlhadlaq HA, Akhtar MJ, Ahamed M: Different cytotoxic and apoptotic responses of MCF-7 and HT1080 cells to MnO 2 nanoparticles are based on similar mode of action. Toxicology. 2019; 411: 71–80. PubMed Abstract \| Publisher Full Text\n\nKusugami K, Fukatsu A, Tanimoto M, et al.: Elevation of interleukin-6 in inflammatory bowel disease is macrophage- and epithelial cell-dependent. Dig Dis Sci. 1995; 40(5): 949–59. PubMed Abstract \| Publisher Full Text\n\nSuzuki T, Yoshinaga N, Tanabe S: Interleukin-6 (IL-6) regulates claudin-2 expression and tight junction permeability in intestinal epithelium. J Biol Chem. 2011; 286(36): 31263–71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAl-sadi R, Ye D, Boivin M, et al.: Interleukin-6 modulation of intestinal epithelial tight junction permeability is mediated by JNK pathway activation of claudin-2 gene. PLoS One. 2014; 9(3): e85345. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith MR, Fernandes J, Go YM, et al.: Redox dynamics of manganese as a mitochondrial life-death switch. Biochem Biophys Res Commun. 2017; 482(3): 388–398. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaint-Georges-Chaumet Y, Edeas M: Microbiota-mitochondria inter-talk: consequence for microbiota-host interaction. Pathog Dis. 2016; 74(1): ftv096. PubMed Abstract \| Publisher Full Text\n\nWang A, Keita ÅV, Phan V, Phan V, et al.: Targeting mitochondria-derived reactive oxygen species to reduce epithelial barrier dysfunction and colitis. Am J Pathol. 2014; 184(9): 2516–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYiu JHC, Dorweiler B, Woo CW: Interaction between gut microbiota and toll-like receptor: from immunity to metabolism. J Mol Med (Berl). 2017; 95(1): 13–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUematsu S, Fujimoto K, Jang MH, et al.: Regulation of humoral and cellular gut immunity by lamina propria dendritic cells expressing Toll-like receptor 5. Nat Immunol. 2008; 9(7): 769–76. PubMed Abstract \| Publisher Full Text\n\nWannemuehler M: Cytotoxic effects of manganese oxide nanoparticles in combination with microbial components on intestinal epithelial cells. 2020. http://www.doi.org/10.17605/OSF.IO/2RKEA" }	[ { "id": "69336", "date": "25 Aug 2020", "name": "Declan McCole", "expertise": [ "Reviewer Expertise Epithelial barrier function", "Inflammatory Bowel Disease", "Intestinal physiology." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary: This paper characterizes potential toxic effects of manganese oxide nanoparticles on intestinal epithelial cells. As the premise for this study, the authors make an important case that the increasing use of MnO nanoparticles may have unpredicted effects on intestinal epithelial barrier integrity. The authors found that manganese oxide nanoparticles (MnO NP) alone or in combination with lysates of the human inflammatory bowel disease (IBD)-associated E. coli, LF82, inhibited monolayer scratch wound healing of the mouse intestinal epithelial MODE-K cell line, enhanced the secretion of interleukin 6 (IL-6), and induced mitochondrial dysfunction. There are concerns with the use of only one cell line as well as the choice of a mouse epithelial cell line for studies involving challenge with a human isolated bacterium. Another substantive concern relates to the lack of a mechanistic connection between the key biological finding – delayed wound healing – and the observations of mitochondrial dysfunction. The paper addresses an important topic and could be significantly enhanced by some straight forward revisions.\n\nComments to the Authors: The major biological finding of the paper with respect to a functional effect of MnO-np on the epithelial barrier paper relates to the delay in wound recovery shown in Figure 3. The other figures focus on mitochondrial effects of MnO-np while one figure shows an increase in IL-6 secretion. The paper is rather disjointed in that a clear mechanistic role for altered mitochondrial function, or the increase in IL-6 secretion, in the delayed wound recovery is not investigated. Testing the effects of a mitochondrial focused antioxidant as performed by other groups i.e. (Wang et al. 2014)1, would better integrate the observational findings of altered mitochondrial function with a potential role in delayed wound healing.\nIt is also unclear as to the significance of the increase in IL-6 secretion relative to the delay in wound healing or mitochondrial dysfunction.\nThe flow of the paper would likely be more compelling if the key biological finding – delayed wound healing – was presented first and was then followed by the analyses of mitochondrial function and cytokine secretion.\nIt is puzzling as to why the authors performed this study with a mouse intestinal epithelial cell line vs. a human IEC line given that their choice of bacteria is a human rather than mouse-derived E. coli. The study also needs to be replicated with an additional cell line to confirm that at least some of the major observed effects are not cell-line specific phenomena (not all of the experiments need to be repeated with an additional cell line).\nA more biologically relevant technical approach to the analysis of cytokine secretion would involve the use of polarized IEC monolayers cultured on transwells so that cytokine secretion from the basolateral surface of the IEC monolayer could be measured. Culture of IEC monolayers on transwells would also allow a more formal investigation of the effects of MnO-np on epithelial barrier properties, an effect that is implied in the Discussion, but no actual data are provided to support this interpretation.\nInclusion of the MnO dose response effect on cell viability would help the reader to understand the relationship of MnO concentration to cell integrity.\nThe use of ROS inhibitors/scavengers would help to validate the suggestion that despite no significant increase in mitochondrial ROS production (Fig 1), that ROS are likely involved in the biological effects of MnO-np observed in MODE-K cells.\nFigure legends 2,3,5-8 should clearly state what treatment comparisons are designated by the individual letters denoting statistical significance.\n\nMinor comments: In the final sentence of the description of data in Figure 1 (page 6), “this data” should be changed to ‘these data’.\nIn the description of data in Figure 2 (page 6), the sentence “However, a significant in IL-6 secretion was detected” should be changed to a ‘However, a significant increase in IL-6 secretion…’\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "76801", "date": "25 Jan 2021", "name": "Tiina Titma", "expertise": [ "Reviewer Expertise Analytical biochemistry", "nanoparticle characterisation and use in medicine", "toxicology", "medicine." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper \"Cytotoxic effects of manganese oxide nanoparticles in combination with microbial components on intestinal epithelial cells” evaluates the effect of exposition of manganese oxide nanoparticles and/or bacterial lysate on mouse intestinal cell line. The aim and subject of the paper carry a significant value as this particular metal is used in medicine in various modes (with at least five different oxidation numbers) and forms, as well in the industry. It is known that excessive exposition of manganese leads to Parkinson like symptoms.\nThe study is well structured, and the methods are adequate and related to cell-layer functions if kept in mind that the intestinal cells form intact epithelial cell monolayer on the basal lamina. It is relevant to use natural exposition site cell models located in airways or digestive tract as mentioned by authors. The permeability of the cell layer could be measured more precisely using transepithelial electrical resistance (TEER) assay as in reference 11.\nThe language is good and text is easy to read.\nIn the section “MTS cell viability assay” there is no mention what kind of readings were taken at 490 and 670 nm. Should it be the same as mentioned in the section “Cytokine release analysis”? The same question applies to the number of independent experiments and replicates per treatment?\nCould the authors kindly explain why the number of independent experiments was two as the internationally validated methods (EURL-ECVAM, ICCVAM) for in vitro assays require at least three independent experiments?\nIt would be beneficial to show the concentration of pure Mn in the solution of 10 μg/mL MnO NP as it makes it easier to compare the results of relevant sources.\nThank you for the nice 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/9-975
https://f1000research.com/articles/9-687/v1	08 Jul 20	{ "type": "Case Report", "title": "Case Report: Cryptococcal meningitis in Hodgkin’s Lymphoma patient receiving brentuximab-vedotin therapy", "authors": [ "Tatiana Cunha Pereira", "Rita Rb-Silva", "Rita Félix Soares", "Nelson Domingues", "José Mariz", "Rita Rb-Silva", "Rita Félix Soares", "Nelson Domingues", "José Mariz" ], "abstract": "Cryptococcus neoformans infections occur mostly in immunodeficient individuals, being the most common opportunistic fungi central nervous system (CNS) infection in HIV seropositive patients. Moreover, other conditions affecting host immunity, such as hematologic malignancies, organ transplantation and immunosuppressive drugs are implicated as risk factors. The authors present a case of a 48-year-old male with Hodgkin Lymphoma for 26 years and submitted to several lines of treatment, diagnosed with cryptococcal meningitis while on therapy with brentuximab. The patient presented with positive cerebral spinal fluid (CSF) cryptococcal antigen plus positive blood cultures. He was put under induction antifungal treatment with liposomal amphotericin B and flucytosine, as well as corticotherapy with dexamethasone with headache improvement and a favorable clinical evolution. There are no reported cases of cryptococcal meningoencephalitis under CD30-directed monoclonal antibody. Furthermore, this case illustrates the risk of Cryptococcus neoformans infection in immunocompromising conditions other than HIV, underlining the need of considering this differential diagnosis when physicians face an opportunist neuroinfection.", "keywords": [ "C. neoformans", "Brentuximab-vedotin", "Hodgkin Lymphoma", "Meningitis" ], "content": "Learning points\n\nCryptococcal meningitis is a common opportunistic central nervous system (CNS) infection among HIV-positive patients. However, these fungi neuroinfection affects HIV seronegative patients.\n\nEvery immunocompromising condition must be assessed and considered a risk factor for an opportunistic fungal meningoencephalitis. A therapeutic agent affecting host immunity, such as with CD30-directed monoclonal antibody, may predispose to opportunistic infections.\n\nCryptococcal meningitis diagnosis may be challenging in cases presenting negative cerebral spinal fluid (CSF) cultures, but cryptococcal polysaccharide antigen titers in CSF correlate with fungal burden.\n\n\nBackground\n\nCryptococcus species have a major predilection for the lungs with potential to spread further, mainly through continuity or through hematogenic and lymphoid pathways, with possible penetration into the blood-brain barrier and CNS involvement1–4.\n\nCryptococcus neoformans infections occur mostly in immunodeficient individuals, being the most common opportunistic CNS infection in HIV-positive patients, counting up to 1 million new infections annually worldwide3,4. It also occurs in transplant recipients, patients with hematological malignancies, as well as patients receiving immunosuppressive medications1,2,4.\n\nThis case reports an opportunistic CNS infection in a patient with Hodgkin Lymphoma under brentuximab-vedotin after multiple lines of treatment for over 20 years, including an allogenic stem cell transplantation. Despite being reported as a common fungal infection in HIV-patients, neuroinfections in patients under CD30-directed monoclonal antibody therapy or other drugs besides immunosuppressants are a rare occurrence.\n\n\nCase presentation\n\nA 48-year-old Caucasian male presented at the outpatient clinic in May 2019 with holocranial headache, more intense at occipital level, lasting for 6 days, with increasing intensity over the last couple hours, associated with photophobia and vomiting.\n\nThe patient was diagnosed in 1993 with Classic Hodgkin Lymphoma, nodular sclerosis subtype, stage IVB, achieving complete remission after first line chemotherapy. Since then, the patient suffered several relapses and underwent radiotherapy, one autologous bone marrow transplant in 1998, as well as an allogenic stem cell transplant in 2001, followed by several lines of chemotherapy. From October 2018 to the admission at the outpatient clinic, the patient was taking Brentuximab due to a hepatic hilar lesion. Throughout image assessments, a large left infratentorial arachnoid cystic lesion was being monitored. (Figure 1).\n\nAt first evaluation, the patient was conscious and aware, hemodynamic stable and subfebrile, presenting general tremors and limited cervical mobility.\n\nBlood workup revealed elevated C-reactive protein with 73.2 mg/L (normal range under 5 mg/L), without other abnormalities.\n\nA head computed tomography (CT) scan showed the pre-existing cystic lesion in the left cerebellopontine angle with a slight right brainstem deviation, without associated edema (Figure 2A), confirmed by magnetic resonance imaging (Figure 2B). The case was discussed with the Neurosurgery Department and a lumbar puncture was postponed as it was considered a high-risk procedure. The patient started antibiotics with ceftriaxone (2 g q12h) and ampicillin. (2g q4h) At day 4, blood cultures came back positive for Cryptococcus neoformans sensitive to Posaconazole, Amphotericin B and Itraconazole, so that patient started Liposomal Amphotericin B (3mg/kg id) and Flucytosine (100 mg/kg per day orally in four divided doses) for 14 days and low dose corticotherapy (4 mg per day). There was a progressive improvement of the symptoms and patient was discharged after 19 days with prescription of Fluconazole (400mg per day).\n\nHead computed tomography (CT) scan showed the pre-existing cystic lesion in the left cerebellopontine angle with a slight right brainstem deviation, without associated edema (2A), as confirmed by magnetic resonance imagining (MRI) (2B).\n\nAfter one month of treatment, a ventricular puncture was performed and normal pressure cerebrospinal fluid (CSF) revealed glucose consumption and elevated levels of proteins (Table 1), as well as positivity for cryptococcal polysaccharide capsular antigen. Follow-up lumbar punctures were performed to assess CSF characteristics and cryptococcal antigen assessment. Patient was kept under consolidation therapy with Fluconazole for 10 weeks with a favorable clinical evolution, as well as decreasing levels of protein and nucleated cells count as seen in Table 1. Patient maintains close surveillance under regular appointments at the Onco-Haematology Clinic. However, headache complaints increased in intensity shortly after dexamethasone discontinuation with an intermittent pattern. Patient died in another hospital about 8 months after the meningitis diagnosis due to a cardiovascular event.\n\nCSF – Cerebrospinal fluid. LP – Lumbar puncture. NV – Normal value.\n\n\nDiscussion\n\nCryptococcal meningitis accounts for up to 1 million new infections annually, mainly affecting HIV-positive patients. Other immunocompromising conditions such as organ transplantation, hematologic malignancies and immunosuppressive drugs constitutes other relevant risk factors to these opportunistic fungi CNS infections1–4.\n\nIn a recent review of Cryptococcus neoformans infections in patients with cancer, 82% corresponded to patients with haematological malignancies and from these patients, approximately 54% had lymphoma5.\n\nThe patient presented several conditions affecting host immunity due to several previous lines of treatment for over 25 years. However, Cryptococcus species were not considered the etiological agent for a CNS infection, emphasizing the need for an initial lumbar puncture to exclude fungal agents when approaching opportunist neuroinfection. This was not possible at first evaluation in this case which delayed antifungal therapy.\n\nAlthough there are many published case reports of Cryptococcosis in patients with lymphoma, this is the first reported case of Cryptococcal neuroinfection in a patient with Hodgkin’s Lymphoma under CD-30-directed monoclonal antibody therapy.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient prior to their death.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nLi SS, Mody CH: Cryptococcus. Proc Am Thorac Soc. 2010; 7(3): 186–96. PubMed Abstract \| Publisher Full Text\n\nBeardsley J, Sorrell TC, Chen SCA: Central nervous system cryptococcal infections in non-HIV infected patients. J Fungi (Basel). 2019; 5(3): 71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGóralska K, Blaszkowska J, Dzikowiec M: Neuroinfections caused by fungi. Infection. [Internet]. 2018; 46(4): 443–59. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMazlarz EK, Perfect JR: Cryptococcosis. Infect Dis Clin North Am. 2016; 30(1): 179–206. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchmalzle SA, Buchwald UK, Gilliam BL, et al.: Cryptococcus neoformans infection in malignancy. Mycoses. 2016; 59(9): 542–52. PubMed Abstract \| Publisher Full Text" }	[ { "id": "66704", "date": "20 Jul 2020", "name": "Adriana Roque", "expertise": [ "Reviewer Expertise Hematology", "Hematology-oncology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI think that this is an interesting report that claims for attention to rare infections in patients under immunotherapy and other novel therapies, especially when they are more difficult to diagnose (due to the location and the infectious agent). This report provides a concise and informative history of the case, mainly focus on the difficulties of the process. I consider that it would be important to provide information about the previous therapeutics that the patient had received, including the transplantation conditioning regimen, as well as the development of graft versus host disease (GvHD) and GvHD therapeutics, because it can help to explain the subjacent immunosuppression state. Another helpful information is to understand if Hodgkin lymphoma was under control at the time of CNS infection presentation.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? 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": "68025", "date": "30 Jul 2020", "name": "Sarah A. Schmalzle", "expertise": [ "Reviewer Expertise Infectious disease", "HIV", "cryptococcosis", "Group A Strep" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 clinically relevant case report as it is purportedly the first report in a patient being treated with a particular immunosuppressive monoclonal Ab.\n\nThere are several minor language and grammatical improvements to be made that will strengthen and clarify the report. It should have an additional round of editing by a native English speaker. Examples:\n\nHIV-positive should be 'people living with HIV'.\n\nAbstract: 'fungi central nervous system..' should be fungal.\n\nBrentuximab should be in lower case throughout. It is not explained with it is 'brentuximab-vedotin' in keywords but 'brentuximab' elsewhere.\n\nEnd of abstract - opportunist should be opportunistic.\n\nThis sentence needs an English language revision: \"However, these fungi neuroinfection affects HIV seronegative patients.\"\n\nBackground - it is not accurate to say crypto has a predilection for the lungs. The lungs are the route of entry, but this is a neurotropic pathogen.\n\n'penetration into' should be 'penetration through'.\n\nCase presentation: \"over the last couple hours\" should be \"over the last couple of hours\" or \"over the last several hours\".\n\nThe use of 'under' to describe being prescribed a treatment is not commonly how this is stated in the US. Try \"treated with\" or \"on treatment for ___ with ___\".\n\nAlso not common to say patients are admitted at the outpatient clinic. Patients are seen or evaluated at a clinic, but admitted to a hospital.\n\nThis sentence also needs to be revised for proper grammar/syntax. \"Throughout image assessments, a large left infratentorial arachnoid cystic lesion was being monitored.\".\n\nSusceptibilities are not commonly reported in Crypto case reports. There is not known resistance and generally all therapy is empiric.\n\nCorticotherapy should be corticosteroid therapy.\n\nThis needs to be reworded for clarity: \"However, Cryptococcus species were not considered the etiological agent for a CNS infection, emphasizing the need for an initial lumbar puncture to exclude fungal agents when approaching opportunist neuroinfection. This was not possible at first evaluation in this case which delayed antifungal therapy.\"\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-687
https://f1000research.com/articles/9-649/v1	26 Jun 20	{ "type": "Research Article", "title": "Open Access of COVID-19-related publications in the first quarter of 2020: a preliminary study based in PubMed", "authors": [ "Olatz Arrizabalaga", "David Otaegui", "Itziar Vergara", "Julio Arrizabalaga", "Eva Méndez", "David Otaegui", "Itziar Vergara", "Julio Arrizabalaga", "Eva Méndez" ], "abstract": "Background: The COVID-19 outbreak has made funders, researchers and publishers agree to have research publications, as well as other research outputs, such as data, become openly available. In this extraordinary research context of the SARS CoV-2 pandemic, publishers are announcing that their coronavirus-related articles will be made immediately accessible in appropriate open repositories, like PubMed Central, agreeing upon funders’ and researchers’ instigation. Methods: This work uses Unpaywall, OpenRefine and PubMed to analyse the level of openness of articles about COVID-19, published during the first quarter of 2020. It also analyses Open Access (OA) articles published about previous coronavirus (SARS CoV-1 and MERS CoV) as a means of comparison. Results: A total of 5,611 COVID-19-related articles were analysed from PubMed. This is a much higher amount for a period of 4 months compared to those found for SARS CoV-1 and MERS during the first year of their first outbreaks (335 and 116 articles, respectively). Regarding the levels of openness, 88.8% of the SARS CoV-2 papers are freely available; similar rates were found for the other coronaviruses. Deeper analysis showed that (i) 67.4% of articles belong to an undefined Bronze category; (ii) 76.4% of all OA papers don’t carry any license, followed by 10.4% which display restricted licensing. These patterns were found to be repeated in the three most frequent publishers: Elsevier, Springer and Wiley. Conclusions: Our results suggest that, although scientific production is much higher than during previous epidemics and is open, there is a caveat to this opening, characterized by the absence of fundamental elements and values on which Open Science is based, such as licensing.", "keywords": [ "Open Access", "Publishing", "Pandemic", "COVID-19", "Scholarly communication", "PubMed", "OA analysis." ], "content": "Introduction\n\nIn the last four months (January–April 2020), due to the COVID-19 pandemic, funders1,2, researchers and publishers (such as Springer or Wiley) seem to agree upon making research outcomes related to the SARS CoV-2 pandemic openly available, including research papers (from preprints - MedRxiv and bioRxiv - to different mechanisms for waiving Article Processing Charges (APCs) or new specific Open Research platforms, as Elsevier or The Lancet). However, traditional practices for scholarly publishing and regular practices to access scientific content might not be mature enough for this massive open endeavour.\n\nThroughout history, research and innovation have been key in the transformation of our society. It has been observed that, in addition to a direct economic benefit, only those societies with a certain level of scientific culture have the capacity to face new risks and participate in new ethical dilemmas, like the ones that we are currently facing. The more scientifically educated societies are, the freer they become, since answers to big social challenges arise from this interaction3. Open Access (OA)/Open Science has been promoted over the last few decades by different stakeholders of the scientific system to make publications openly accessible, and more recently, also data and other research outcomes, in order to make them FAIR (Findable, Accessible, Interoperable and Reusable). All these initiatives aim to boost a democratic scientific advance in which scientists but also citizens are involved.\n\nIn the current situation of a global pandemic, OA becomes urgent. The emergence of the virus that causes the disease known as COVID-19 first reported by the Chinese authorities in late December 2019, has resulted in an unprecedented level of collaboration among researchers around the world4–6. A health crisis, such as the SARS CoV-2 pandemic, requires special effort and collaboration within the scientific community in order to generate and disseminate new results, while trying to avoid duplication of efforts globally.\n\nIn this unique context of the pandemic, publishers are announcing massive OA changes, primarily by making their coronavirus-related articles freely available through databases, such as PubMed Central, together with other public repositories. SPARC Europe stated that overnight COVID-19 heightens the need for Open Science, and we cannot agree more. But we wonder if this openness might be enough in such a demanding and urgent episode for Science, and coincidently we wonder if the scientific community is ready to share and consume openly such information. This work aims to make an initial analysis of scientific production concerning COVID-19 and its level of openness as a first step to assess the current research publication model and the unpredicted outcome of openness of research in this global health emergency. Thus, this paper analysed all scientific content openly available from PubMed database.\n\n\nMethods\n\nIn order to analyse publications concerning COVID-19 and their level of openness, we have chosen PubMed instead of other multidisciplinary databases, like Web of Science (WoS) or Scopus. PubMed is one database developed by the National Center for Biotechnology Information (NCBI) at the National Library of Medicine (NLM) in the USA. It is one of the most used databases to find biomedical scientific content. This database gathers over 14 million bibliographic citations and it provides access to MEDLINE articles and PubMed Central (PMC), an extensive digital repository created in 2000 for biomedical and life sciences Open Access publications. Unlike many other research databases, such as WoS, PubMed also includes articles that are “in process”; this means a status prior to being indexed with MeSH terms, and articles submitted by publishers as pre-prints (i.e. articles that haven't gone through peer review)7. This aspect is crucial for this study since, at this moment, scientific papers are being published very fast and may not have yet undergone peer review8.\n\nSince during the global pandemic period, the scientific community is posting articles that are freely accessible through the NCBI, data were collected from the PubMed database in order to analyse every COVID-19-related scientific paper that is currently published (including PMC)9. In an attempt to evaluate the most accurate list of publications, we exported all results obtained from the suggested search queries offered by NLM (NCBI webpage), as follows: “2019-nCoV OR 2019nCoV OR COVID-19 OR SARS-CoV-2 OR (wuhan AND coronavirus)”. Only articles published from January 1st to April 23rd of 2020 were considered. No exclusions were made in the type of article (journal article, books, reviews, clinical trial or meta-analysis) or in the language, choosing in each case every article offered by PubMed.\n\nIn line with the objective of analysing published papers during other emergency circumstances, similar search procedures were applied to the SARS CoV-1 pandemic (query: “SARS CoV” OR “Severe Acute Respiratory Syndrome Coronavirus”; period searched: from 2003 to 2006) and MERS CoV epidemic (query: “MERS CoV” OR “Middle East Respiratory Syndrome Coronavirus”; period searched: from 2013 to 2016).\n\nIn order to determine the effect that this health emergency is having on the availability of the scientific production, we decided to compare it with the availability in a normalized situation, for which we performed the same analysis using two chronic diseases: low grade glioma (query: “low grade glioma”) and peptic ulcer (query: “peptic ulcer”), which, as seen by our search, have stable publication patterns for the last three years (2017 to 2019).\n\nObtained results, without exclusion, were exported and uploaded to OpenRefine, a free open source tool that helps exploration of large data sets, and has the capability to link and extend these data sets with different webservices. In this study, OpenRefine was used to manage data but also as the key element in order to link our PubMed data set with Unpaywall, the selected tool for analysing the OA content of all these data. Unpaywall (previously known as oaDOI) is a database introduced in 2016 as a service to check OA availability of journal articles identified by their Digital Object Identifier (DOI)10. Unpaywall is currently used more than 50,000 times a day and is maintained by Our Research, a non-profit company previously called Impactstory11. It offers access to the OA status of scientific journals, through an open application programming interface (API). Unpaywall also shows license information and variable version availability from different repositories10,11.\n\nWoS, which includes OA information from Unpaywall12,13, classifies OA papers in five-categories that we consider in this work: Gold, OA journal indexed by the Directory of Open Access Journals (DOAJ); Hybrid, subscription-based journals including some OA articles; Green, toll-access on the publisher page, but there is a free copy in an OA repository; and Bronze, articles freely available on websites hosted by their publisher, either immediately or following an embargo, but are not formally licensed for reuse14. Unpaywall also provides information about Creative Commons (CC) licensing of each document (commonly Gold OA or Hybrid journals). Copyright licenses, released by Creative Commons, are variable and range from more open permissions (CC or CC-BY) to more restrictive ones (CC-BY-ND, CC-BY-NC, CC-BY-NC-ND or CC-BY-NC-SA)15.\n\nArticles from dates other than the ones specified were not considered (even if PubMed includes some out-of-date articles in its results). Only articles with a DOI were considered, and among them, there was a proportion not recognized by Unpaywall and thus, also not considered. Hence, the exclusion criteria after Unpaywall analysis includes out-of-date and those not scanned by Unpaywall (including papers without DOI).\n\nAlso, the Unpaywall system indexes thousands of institutional and subject repositories, but there are some still missing, and the database updates periodically, so some data might have changed.\n\n\nResults\n\nThe data obtained about SARS CoV-2 from January 1st to April 23rd 2020 are shown in Figure 1. In total, 6,223 articles were retrieved from PubMed. Of these 10 were from 2019, 182 did not have a DOI assigned and 485 were not recognized by Unpaywall, and so were excluded from analysis; therefore, analysis was performed on a total of 5,611 articles.\n\n(a) Number of total and OA papers published during SARS CoV-2 pandemic. (b) Percentage of publications divided by their OA publishing mode. (c) Unpaywall-used source to obtain OA papers. (Data extracted from PubMed: 23rd April 2020).\n\nFrom the data, it can be seen that the number of articles published during the selected period increases daily. Figure 1a shows that 88.8% (n=4,986) of articles were published as OA. Regarding the type of OA, 67.4% (n=3,359) are classified as Bronze OA, followed by Gold OA (21.5%), Hybrid journals (7.8%), and Green OA (3.3%) (Figure 1b). All these OA articles (n=4,986) were found by Unpaywall through different sources of information (Figure 1c), mostly (73.8%) as free articles (PDF or HTML). It is worth mentioning that 43% of the OA papers (n=2,414) have a copy in a repository, even if they are Gold, Hybrid or Bronze, which is known as shadowed Green documents14.\n\nIn order to deeply analyse the OA situation, we also reviewed license information of all the OA papers. Figure 2 shows that most of these articles lack a license (76.4%). Most open licenses (CC, CC-BY and Public Domain (PD)) are present in 13% of the papers, while the most restrictive ones (CC-BY-NC, CC-BY-NC-ND, CC-BY-NC-SA, CC-BY-SA and CC-BY-ND) are represented by more than 10% of all the considered papers (Figure 2b). Publisher implied licenses (implied OA) are included as the more restrictive ones. From all licensed papers (n=1,175), 44.3% bear a restricted one. It is remarkable that 258 of the articles classified as Gold OA (24%) don’t bear any license.\n\n(a) Distribution of papers based on license category. Licenses were divided as: CC, CC-BY, PD, Implied OA, CC-BY-NC, CC-BY-NC-ND, CC-BY-NC-ND-SA, CC-BY-ND; and those without any particular license. (b) Distribution of papers with OA license (CC, CC-BY and PD), restricted license (Implied OA, CC-BY-NC, CC-BY-NC-ND, CC-BY-NC-ND-SA, CC-BY-ND) or without a license. (Data extracted from PubMed: 23rd April 2020).\n\nFurthermore, the most frequent publishers and journals during this period in relation to SARS CoV-2 were studied. The most frequent publisher is Elsevier, who published ~30% of papers, followed by Wiley (13.6%) and Springer (10.7%) (Figure 3a). In terms of journals, The British Medical Journal (The BMJ), Journal of Medical Virology and The Lancet are those with the largest number of papers: 4.2, 3.1 and 2.2% of all analysed papers, respectively (Figure 3b).\n\nNumber and percentage of total publications distributed by most frequent publishers (a) and journals (b). (Data extracted from PubMed: 23rd April 2020).\n\nBased on these results, we specifically studied the COVID-19-related articles published by Elsevier, Wiley and Springer (Figure 4). While Elsevier and Springer release almost all SARS CoV-based articles as OA (96.3%), Wiley retains 28.3% as closed access (Figure 4a). All three publishers publish the majority of their papers as Bronze OA (Figure 4b). Note that Elsevier is the only one (out of these three) that classifies more than 2% of its articles as Green OA (n=130; 8.1% of all OA papers). Elsevier has also published approximately 17% (n=274) of these documents as Gold OA, 1.25% and 12.1% more than Springer and Wiley, respectively. Looking at licensing, most of the OA publications from these publishers lack a license, being Springer the one with highest license number (24.3%) (Figure 4c). Regarding specific OA licensing, Springer publishes 89.9% of its licensed articles under CC-BY, Wiley does the same but with less than the half of its collection (44.4%) and Elsevier has the most restrictive conditions: 89.5% of the licensed papers carry CC-BY-NC-ND licenses (Figure 4d).\n\n(a) Percentage of OA publications of the most relevant publishers: Elsevier, Wiley and Springer. (b) Distribution of their open content by Gold, Hybrid, Green or Bronze status. (c) Distribution of the licensed and non-licensed articles of the three publishers. (d) Distribution of the most frequent licensing type by each publisher: from the most restrictive licenses to the more open ones. (Data extracted from PubMed: 23rd April 2020).\n\nIn order to compare the scientific production and OA publication during global health emergencies, both SARS CoV-1 and MERS CoV-related publications were studied using the PubMed database, taking into account the times for the beginning of each outbreak.\n\nIn the case of the SARS CoV-1 (Severe Acute Respiratory Syndrome CoronaVirus-1) epidemic, the first case was discovered in China during November 200216. We therefore analysed publications published in 2003, 2004, 2005 and 2006 (Figure 5). For the period from 2003 to 2006, PubMed returned a total of 2,396 articles, of which, after exclusion criteria, 1,858 were considered (476 lacked DOI, 58 were out-of-date and 4 were not recognized by Unpaywall). There was an increase in the number of publications from 2003 to 2004, with a decline onwards. The percentage of OA publications increased from 80 to 87% in the first year, maintaining a stable average of 84% throughout the analysed period (Figure 5a). Among these open articles, 63.1% were published as Bronze OA, 19.6% as Green OA, 13.9% as Gold OA, and 3,3% as Hybrid journals (Figure 5b). From all the OA papers, almost 88.8% (1,389) lacked a license, including a high proportion (44.5%) of Gold OA papers.\n\n(a) Number of total and OA publications about SARS CoV-1 epidemic during the first 4 years from the start of the epidemic. (b) OA category of the OA published articles. (Data extracted from PubMed: 19th April 2020).\n\nNext we performed the searches for the MERS CoV (Middle East Respiratory Syndrome Coronavirus) epidemic, whose outbreak began in September 2012 in Saudi Arabia17. A total of 1,129 papers were obtained for the specified period (2013 to 2016), of those 78 don’t have any DOI and Unpaywall did not recognize 8, giving as a result a total of 1,043 analysed articles. In this case, this number is significantly lower than the one found for SARS CoV-1 over time. In 2016, the year in which most papers are registered (n=345), the percentage of these published as OA remains constant and is very high, with an average of 93.5% (Figure 6a). Unlike SARS CoV-2 and SARS CoV-1, 44.3% of MERS-related OA publications were published as Gold OA (Figure 6b). From all the OA papers, 61.3% (n=598) lack a license, an important proportion corresponding to Gold OA papers (29.4% of Gold).\n\n(a) Number of total and OA publications based on the MERS CoV epidemic during the first 4 years from the epidemic outbreak. (b) OA category of the OA published articles. (Data extracted from PubMed: 19th April 2020).\n\nIn order to determine if these results are a consequence of the current extraordinary circumstances, a control of the research was established through the analysis of open content of chronic diseases considered constant over time. We performed searches for “low grade glioma” and “peptic ulcer”, which harbour similar output levels compared to SARS CoV-1 and MERS, obtaining a constant OA proportion for each case over the last 3 years (Figure 7). This rate is low for all cases, with an average of 55.1% and 51.5% for low grade glioma (Figure 7a) and peptic ulcer (Figure 7b), respectively. In addition, articles concerning both diseases were mostly published as Gold OA (Figure 7a and 7b). In these two cases, the number of OA articles without a license represents around 40%.\n\nNumber of publications, OA percentage and category of articles related to low grade glioma (a) and peptic ulcer (b) during 2017, 2018 and 2019. (Data extracted from PubMed: 20th April 2020).\n\n\nDiscussion and conclusion\n\nCompared to other emergency crises such as, SARS CoV-1 or MERS CoV epidemics, the number of published papers during the current COVID-19 pandemic is huge. Our study (based only on the PubMed database) reveals that in only four months, the number of these articles is 17-times more than the number of documents available in the first year in the case of SARS CoV-1, and 48-times in the case of MERS CoV. Shortening of acceptance rates by journals is giving rise to information overload both for the scientific community but also for society, making it difficult to ascertain what really has a significant scientific value and as a consequence may affect decision-making.\n\nIn addition to the massive scientific production, after the pandemic declaration, publishers have made, not only COVID-19 but also previous SARS CoV-1 and MERS CoV related papers, openly available. From our study, both SARS-like viruses share the same limited conditions, i.e. are non-licensed Bronze OA articles. On the contrary, a large number of MERS CoV-related papers present as Gold OA, suggesting high public funding from funders with OA policies during this period. In this context, it is surprising that there is a large number of Gold OA articles without licenses for all three diseases, which raises some uncertainties about whether some journals should still be listed in the DOAJ.\n\nWhile Gold OA makes papers available immediately by the publishing journal itself, the predominant Bronze OA category, found by the present study, means that papers are freely hosted on publisher websites, without a license at all. Little is discussed in the OA literature about this category, but what is clear is that articles under this group without a categorised license do not allow extended reuse rights beyond reading. Thus, this “open” label removes rights to share or redistribute and, moreover, the publisher can revoke this access at any time. For instance, publishers’ announcements about their temporary fee drop on coronavirus-related research is limited only to the duration of the crisis (Springer Nature or Elsevier).\n\nIn line with this, this study found that PubMed-hosted COVID-19 papers that have a copy included in a repository almost reach 50% of OA papers; however, only 3% are assigned under Green OA status. This implies that many of the Bronze OA articles - around 60% - have a copy in the repositories searched by Unpaywall, which can be removed upon publisher request.\n\nAnother point to highlight, as defined by Piwowar et al.14, is the fact that many of these Bronze OA publications have been published in Hybrid journals. These papers, due to their accessibility, benefit from greater citation. It is not surprising that during this emergency situation, they are attracting the attention and curiosity of the entire world, including not only the scientific community but also non-scientific, increasing the citations and so the journals’ reputation. After publishers decide to reinstate paywalls, as the majority of the documentation is not free all the time, the number of subscriptions might be affected, since it is possible that new non-subscribed readers obtained during this pandemic period have read articles from these journals and want to continue doing it.\n\nWhat is most interesting about the effect of the COVID-19 emergency on scientific research disclosure is what it says about the current publication model: it fails when a critical need arises for fast data dissemination. Our analysis demonstrates that the current alternative that is in use falls short of expectations of being the best model, since this fast opening lacks basic OA principles, which are required in order to be transparent, reusable and good for the society. This could also have an important impact on a possible scenario where new outbreaks occur in the coming months or years.\n\nWe finally conclude that it seems clear that all stakeholders agree that Science only works when knowledge is shared. This unique and exceptional pandemic situation gives the opportunity to analyse the current publishing system in order to start doing things in a way that benefits the whole community, both researchers and society at large. This study has presented a part of Open Science-related issues and hopefully stimulates further research from the OA community regarding the use of Bronze OA and Hybrid journals.\n\n\nData availability\n\nZenodo: Open Access of COVID-19 related publications in the first quarter of 2020: a preliminary study based in PubMed, http://doi.org/10.5281/zenodo.382603817.\n\nThis project contains the following underlying data:\n\n- Excel datafile with Unpaywall analysis of each research query.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nDimity Flanagan (Manager, Scholarly Communications, University of Melbourne) for her review and valuable suggestions.\n\n\nReferences\n\nCoronavirus Open Acess Letter. Accessed May 5, 2020. Reference Source\n\nBlair C: Request for Information: Public Access to Peer-Reviewed Scholarly Publications, Data and Code Resulting From Federally Funded Research. 2020. Reference Source\n\nUNESCO - United Nations Educational Scientific and Cultural Organization: Science for Society. Accessed May 24, 2020. Reference Source\n\nShanmugaraj B, Siriwattananon K, Wangkanont K, et al.: Perspectives on monoclonal antibody therapy as potential therapeutic intervention for Coronavirus disease-19 (COVID-19). Asian Pac J Allergy Immunol. 2020; 38(1): 10–18. PubMed Abstract \| Publisher Full Text\n\nRothan HA, Byrareddy SN: The epidemiology and pathogenesis of coronavirus disease (COVID-19) outbreak. J Autoimmun. 2020; 109: 102433. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAhn DG, Shin HJ, Kim MH, et al.: Current status of epidemiology, diagnosis, therapeutics, and vaccines for novel coronavirus disease 2019 (COVID-19). J Microbiol Biotechnol. 2020; 30(3): 313–324. PubMed Abstract \| Publisher Full Text\n\nFalagas ME, Pitsouni EI, Malietzis GA, et al.: Comparison of PubMed, Scopus, Web of Science, and Google Scholar: strengths and weaknesses. FASEB J. 2008; 22(2): 338–342. PubMed Abstract \| Publisher Full Text\n\nTorres-Salinas D: Ritmo de crecimiento diario de la producción científica sobre Covid-19. Análisis en bases de datos y repositorios en acceso abierto. El Prof la Inf. 2020; 29(2). Publisher Full Text\n\nHe J, Li K: How comprehensive is the PubMed Central Open Access full-text database? In: IConference 2019 Proceedings. iSchools; 2019. Publisher Full Text\n\nElse H: How Unpaywall is transforming open science. Nature. 2018; 560(7718): 290–291. PubMed Abstract \| Publisher Full Text\n\nSingh Chawla D: Half of papers searched for online are free to read. Nature. 2017. Publisher Full Text\n\nBosman J, Kramer B: Open access levels: a quantitative exploration using Web of Science and oaDOI data. PeerJ Preprints. 2018; 6: e3520v1. Publisher Full Text\n\nWeb of Science Core Collection Help. Accessed May 9, 2020. Reference Source\n\nPiwowar H, Priem J, Larivière V, et al.: The state of OA: A large-scale analysis of the prevalence and impact of Open Access articles. PeerJ. 2018; 6: e4375. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCreative Commons: Creative commons license spectrum.svg - Wikimedia Commons. 2016; Accessed May 5, 2020. Reference Source\n\nCleri DJ, Ricketti AJ, Vernaleo JR: Severe Acute Respiratory Syndrome (SARS). Infect Dis Clin North Am. 2010; 24(1): 175–202. Publisher Full Text\n\nZaki AM, Van Boheemen S, Bestebroer TM, et al.: Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med. 2012; 367(19): 1814–1820. PubMed Abstract \| Publisher Full Text" }	[ { "id": "65639", "date": "06 Jul 2020", "name": "Cameron Neylon", "expertise": [ "Reviewer Expertise research evaluation", "open access 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\nReview and Replication Report for Arrizabalaga et al. (2020)\nGeneral Observations\nThe paper addresses an important issue on a dynamic and moving subject, the availability of research on COVID-19 within the context of the pandemic. This is a useful and potentially important record of the state of the literature at a particular point in time. Its timeliness is also related to some of its weaknesses in terms of how the state of the relevant literature is changing. Nonetheless, it presents a useful record and, with some relatively minor alterations, will provide an important record of a moment in time.\nRecommendations for clarification\nThere are a series of changes and clarifications I would recommend to the paper as the conclusions depend on the specificity of categories of open access referred to. It is important to be clear about the details of what is meant by categories such as 'hybrid' and 'bronze' and how closely these relate to the heuristics that are used to detect them, which are necessarily imperfect.\nSpecifically, it is important to distinguish between the category of articles that are temporarily released by publishers from behind a paywall, and those articles that are detected by a process of identifying free copies on a publisher website without an explicit license ('bronze'). As the argument of the paper hinges on the identification and categorisation of these articles and implicitly on the motivations of publishers in releasing them it is critically important that the category of access models (promotional or emergency release) is distinguished from the categories that can be detected ('bronze').\nSpecific suggested changes to address these and related issues:\nUnder 'Data analysis' it is not immediately clear to me why the Web of Science classification is referred to. I would argue that what should be presented is the detailed implementation of exactly how the categories are assigned in this article (see Replication report below for an example of this). If the categories provided by Unpaywall are used directly this should be explained.\n\nMore detail on the process of data preparation would be helpful. The provision of the finalised data is very useful but details of how the Unpaywall data was collected (via the API in OpenRefine or by upload of a set of DOIs?) and exactly when (because this makes a difference to analysis, see below).\n\nThroughout the discussion, there is a potential for confusion with terms like 'non-licensed Bronze'. I would use 'Bronze' throughout, perhaps repeating the point that it is by definition non-licensed. Similarly the statement '...many of these Bronze OA publications have been published in Hybrid journals...' is confusing as by the definitions used here Bronze will always be in a hybrid journal.\n\nA related issue is that I would prefer to explicitly use a term like 'DOAJ Gold' to refer to articles in purely open access journals as there is significant variation across the literature in the application of this term and being explicit throughout would help.\nThere is also some confusion in the description of Green OA. Specifically, the definition of Green adopted here is one that applies only to those articles that are not also Gold. This is standard practice, although I personally think it inadvisable, here it leads to significant confusion. In fact, the contribution of repository access to this corpus is nearly as great as that of publishers with 43% being described as \"shadowed green\". I would argue for a more detailed analysis of the repositories being used in the results section.\nIn policy and analysis terms this is arguably as important a contribution to access as that of publishers. I would argue for a greater analysis of this part of the corpus (see replication report for further details). The choice of Pubmed Central to accept the deposit of articles with no guarantee of long-term access is a significant potential issue. This both raises questions about definitions of \"green\" open access and licensing that deserve a little more attention in the discussion in my view.\nThe paragraph in the discussion that commences \"In line with this...\" is difficult to parse. It is not clear to me that the lack of a license on the publisher site (which results in categorisation as bronze) necessarily flows through to the licensing of the Pubmed Central version. This deserves further analysis (see below). The paragraph reads as though the assignment of only 3% to green implies that the repository copies are not guaranteed. My reading of the methodology does not agree with this. This strengthens the argument for an explicit description of the category assignment.\nFinally, I think the conclusion is probably too strong on what the analysis demonstrates vs what the concerns of the authors are. While I agree with their conclusion that it is unfortunate that the release of otherwise restricted content in the context of the pandemic has such limitations in terms of the time frame and re-use this analysis cannot show the downstream effects of those restrictions, which will need to await future analysis. I think a sharper distinction between the observations made and the concerns of the authors would benefit the article.\nMinor issues\nFigure 1 has a number of misleading characteristics. In Figure 1a a bar chart is presented that shows both all articles and the oa subset but adding the two together. Figure 1c is also confusing. As noted below I don't understand why the data has been divided up the way it has. Both the conflation of the two evidence types for which Unpaywall found free articles, combined with leaving out of DOAJ as evidence source for the second pie chart seems odd and these results are not used elsewhere in the paper. I would leave 1c out and use a Venn diagram for 1a and a bar chart for 1b Figure 2 and related text. The license category of 'cc' is presumably cc0.\nI find Figure 4d confusing. Would it not be better to show some quantitative parameter for each of the publishers rather than the -OPEN and +OPEN? Perhaps open licenses as a proportion of all articles or something similar?\nIn analysing past outbreaks the issue of increases in repository-mediated (green) OA over time should be explicitly mentioned. This might particularly be included in a comparison of those repositories that are contributing to access. This does not directly affect the conclusions of these sections as the proportion of green is not otherwise interpreted but the potential for confusion means this should be at least mentioned with a statement saying that it is not therefore possible to directly compare the levels of green open access across these outbreaks.\nReplication Report\nI report on a direct replication using the supplied data. Broadly speaking I confirm the overall results with some reservations and slight differences which are noted below. There seems little value in reproducing the Unpaywall data from the supplied DOIs. A manual search of PubMed could be used to confirm the numbers and identity of DOIs but I do not conduct that here at this point.\nThe full code for the Replication report can be found at Github as a Jupyter Notebook at: https://github.com/cameronneylon/replication_report_Arrizabalaga_2020\nAnd on Mybinder.org at: https://mybinder.org/v2/gh/cameronneylon/replication_report_Arrizabalaga_2020/master\nHere I provide only the main points in summary. See Github for the fully worked analysis and code for comparison purposes.\nMinor issues\nThe dataset has 5621 rows of data, not 5611 as specified in the paper. Is this to do with blank entries or entries without DOIs?\n\nThere are 4989 oa articles by my analysis, not 4986 as specified in the paper. Comparison to the provided data provides 4991 oa articles, and the difference is explained by the two entries for which the JSON does not parse.\n\nNot immediately clear why for Fig 1c the two categories of free articles have been combined?\n\nWhy in Figure 1c are the oa types reported only for those articles where the evidence type is either free article or free pdf? Why are the DOAJ evidence examples not included?\n\nFigure 2. Slight variation in the percentages calculated from the dataset.\n\nThere are slight issues in 4b and 4c with the license assignment.\n\nAs noted in general comments I would drop Figure 4d as it is confusing and it is not clear to me that it is supported by the data where Wiley does not appear to have many more open licenses than Elsevier.\n\nMajor Issues\nThe numbers in the paper do not seem to correspond directly to those in the dataset provided\n\nIt appears the article does not use DOAJ as the criterion for gold but the is_oa_journal field from unpaywall. This explains the variance between my analysis and that in the article for \"gold\" as defined by my code (16% vs 19% using the data provided, vs 21.5% given in Figure 1).\n\nIn Figure 5-7 I think there may be an error in the counting of OA articles, counting all those articles for which there is an 'is oa' entry and not only those where it is set to True.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5769", "date": "12 Aug 2020", "name": "Olatz Arrizabalaga", "role": "Author Response", "response": "Dear Cameron,Find here below, in italics, all the explanations to your helpful comments and insights.General ObservationsThe paper addresses an important issue on a dynamic and moving subject, the availability of research on COVID-19 within the context of the pandemic. This is a useful and potentially important record of the state of the literature at a particular point in time. Its timeliness is also related to some of its weaknesses in terms of the how the state of the relevant literature is changing. Nonetheless it presents a useful record and, with some relatively minor alterations, will provide an important record of a moment in time.Thank you very much for your thoughtful overall evaluation. We do agree with this perception and we are addressing all your comments and recommendations in Version 2 of the paper, and we also underline the state of the changing status of the relevant literature about COVID-19 in order to contribute, with this piece, to the meta-research about so relevant topic in the current challenging context. Recommendations for clarification, There are a series of changes and clarifications I would recommend to the paper as the conclusions depend on the specificity of categories of open access referred to. It is important to be clear about the details of what is meant by categories such as 'hybrid' and 'bronze' and how closely these relate to the heuristics that are used to detect them, which are necessarily imperfect. Specifically it is important to distinguish between the category of articles that are temporarily released by publishers from behind a paywall, and those articles that are detected by a process of identifying free copies on a publisher website without an explicit license ('bronze'). As the argument of the paper hinges on the identification and categorisation of these articles and implicitly on the motivations of publishers in releasing them it is critically important that the category of access models (promotional or emergency release) is distinguished from the categories that can be detected ('bronze').The authors agree completely with this perception. In the V2 of the paper we update these conclusions by analysing the licenses of each paper together with each location. In this context, we can take conclusions about whether the publisher intend to contribute to the emergency situation or not (ie. The role of PMC during this global health emergency as the main COVID-19 repository).Specific suggested changes to address this and related issues: Under 'Data analysis' it is not immediately clear to me why the Web of Science classification is referred to. I would argue that what should be presented is the detailed implementation of exactly how the categories are assigned in this article (see Replication report below for an example of this). If the categories provided by Unpaywall are used directly this should be explained. We used WoS classification as it is based on the Unpaywall’s one. But you are right, it is easier to follow by just mentioning the categories provide by Unpaywall. V2 of the paper follows this last one. More detail on the process of data preparation would be helpful. The provision of the finalised data is very useful but details of how the Unpaywall data was collected (via the API in OpenRefine or by upload of a set of DOIs?) and exactly when (because this makes a difference to analysis, see below). We agree upon this and we detail in this sense the description of the methods and tools in V2 of the paper. V2 includes the following points: PubMed was selected as our database after a comparative study performed versus WoS. Even if it would have been easier to perform de study with WoS (as it includes an “open access” filter that PubMed does not), this presented false “closed” articles that at the same time were open in PubMed. It seems that in March, WoS was not updated enough and lacked of OA information. Unpaywall data was collected via the API in OpenRefine. PubMed data was uploaded to OpenRefine and via the API all the Unpaywall data was collected. It is important to mention that Unpaywall has notified us about an update of one of its filters during the timeframe of our study, thus affecting some of our results. This implies some license information that we are updating then the data. The publisher allowed us that update and it is included in V2 of the paper. Throughout the discussion there is a potential for confusion with terms like 'non-licensed Bronze'. I would use 'Bronze' throughout, perhaps repeating the point that it is by definition non-licensed. Similarly the statement '...many of these Bronze OA publications have been published in Hybrid journals...' is confusing as by the definitions used here Bronze will always be in a hybrid journal. You are right, it seems redundant. Also important to point out that in the new analysis there are 31 Bronze papers with licenses in the repositories they are uploaded, which means that there are a few within this category that are not only promotional for the publisher or pure bronze (as you said, by definition non-licensed). A related issue is that I would prefer to explicitly use a term like 'DOAJ Gold' to refer to articles in purely open access journals as there is significant variation across the literature in the application of this term and being explicit throughout would help. This is an important issue when analysing the data. You are right that we should clarify when defining each OA category. Based on Unpaywall Gold definition, “not only DOAJ indexed journals are included, but also 100% OA journals”. Unpaywall clarifies in this sense how they set the Gold OA status of an article (see: https://support.unpaywall.org/support/solutions/articles/44001792752):We set the oa_status of an article to “gold” if that article is published in a fully OA journal. We have three steps to decide if a given journal is fully OA: is in DOAJ. If not: Is it a known fully-OA publisher? We maintain a small whitelist of publishers that we know only publish OA content (for instance, many publishers using the SciELO model). If the journal’s publisher is on this list, it’s a fully OA journal, even though it’s not in DOAJ. Does the journal publish only OA articles? Since we index the complete output of over 70,000 journals, we’re able to check our database to see if a given journal publishes exclusively OA content. If they do, they’re a fully OA journal, even if they’re not listed in DOAJ. So, Gold OA category includes DOAJ indexed journals, but also other 100% OA considered ones that UnpayWall is getting in its database. So we do not use DOAJ Gold but ‘Gold’ taking into account that “Gold” is, at the end, what UnpayWall has as Gold, following the 3 steps cited above.There is also some confusion in the description of Green OA. Specifically, the definition of Green adopted here is one which applies only to those articles that are not also Gold. This is standard practice, although I personally think it inadvisable, but here it leads to significant confusion. In fact, the contribution of repository access to this corpus is nearly as great as that of publishers with 43% being described as \"shadowed green\". I would argue for a more detailed analysis of the repositories being used in the results section.Totally agree, when re-describing OA categories we are going to take this into account as most of the Gold and Hybrid articles present a repository copy. In the new analysis in V2 of the paper, all these repository copies are deeply analysed.In policy and analysis terms this is arguably as important a contribution to access as that of publishers. I would argue for a greater analysis of this part of the corpus (see replication report for further details). The choice of PubMed Central to accept the deposit of articles with no guarantee of long-term access is a significant potential issue. This both raises questions about definitions of \"green\" open access and licensing that deserve a little more attention in the discussion in my view.Yes, together with the previous point, this is carefully addressed and discussed in the new version (V2) of the article.The paragraph in the discussion that commences \"In line with this...\" is difficult to parse. It is not clear to me that the lack of a license on the publisher site (which results in a categorisation as bronze) necessarily flows through to the licensing of the Pubmed Central version. This deserves further analysis (see below). The paragraph reads as though the assignment of only 3% to green implies that the repository copies are not guaranteed. My reading of the methodology does not agree with this. This strengthens the argument for an explicit description of the category assignment.We also agree upon this this point, together with last two points, The further analysis has demonstrated that most of the repository copies don’t carry licenses (well, they call it “custom licenses” as the ones stated by Elsevier or Springer Nature). This highlights the role of PMC.Finally I think the conclusion is probably too strong on what the analysis demonstrates vs what the concerns of the authors are. While I agree with their conclusion that it is unfortunate that the release of otherwise restricted content in the context of the pandemic has such limitations in terms of time frame and re-use this analysis cannot show the downstream effects of those restrictions, which will need to await future analysis. I think a sharper distinction between the observations made and the concerns of the authors would benefit the article.You are right, our strong opinions lead to strong conclusion. We have tried to “relax” them in the new version of the paper.Minor issues Figure 1 has a number of misleading characteristics. In Figure 1a a bar chart is presented that shows both all articles and the oa subset but adding the two together. Figure 1c is also confusing. As noted below I don't understand why the data has been divided up the way it has. Both the conflation of the two evidence types for which Unpaywall found free articles, combined with leaving out of DOAJ as evidence source for the second pie chart seem odd and these results are not used elsewhere in the paper. I would leave 1c out and use a venn diagram for 1a and a bar chart for 1b.Ok, we will change the Figures 1a and 1b, and leave 1c out. The update carried out by Unpaywall reflects the vagueness of this figure, and the new analysis has been done by looking at each evidence (up to 4 locations) of each article. Instead, we can include a Venn diagram overlapping each OA category with the ones with a repository copy.Figure 2 and related text. The license category of 'cc' is presumably cc0.Yes. It is CC0, It is update in the v2 or the paper.I find Figure 4d confusing. Would it not be better to show some quantitative parameter for each of the publishers rather than the -OPEN and +OPEN? Perhaps open licences as a proportion of all articles or something similar?We agree. We have clarified the representation in Figure 4d.In analysing past outbreaks the issue of increases in repository-mediated (green) OA over time should be explicitly mentioned. This might particularly be included in a comparison of those repositories that are contributing to access. This does not directly affect the conclusions of these sections as the proportion of green is not otherwise interpreted but the potential for confusion means this should be at least mentioned with a statement saying that it is not therefore possible to directly compare the levels of green open access across these outbreaks.Totally agree. Issues IdentifiedMinor issues The dataset has 5621 rows of data, not 5611 as specified in the paper. Is this to do with blank entries or entries without DOIs? 8 belong 2019 y 2 contain JSON error, so 10 were excluded. There are 4989 oa articles by my analysis, not 4986 as specified in the paper. Comparison to the provided data provides 4991 oa articles, and the difference is explained by the two entries for which the JSON does not parse. After excluding the previous 10, 4986 is the final number. Not immediately clear why for Fig 1c the two categories of free article have been combined? Why in Figure 1c are the oa types reported only for those articles where the evidence type is either free article or free pdf? Why are the DOAJ evidence examples not included? In order to avoid confusion fig 1c is be taken out in V2 of the paper. Figure 2. Slight variation in the percentages calculated from the dataset. You have performed the analysis for the hole publications, not just for the OA ones. We calculate the percentages only of the OA collection. There are slight issues in 4b and 4c with license assignment. If our Gold definition is used, the numbers should be correct. Even so, these numbers change in V2 of the paper when we consider the figures updated by UnpayWall. As noted in general comments I would drop Figure 4d as it is confusing and it is not clear to me that it is supported by the data where Wiley does not appear to have many more open licenses than Elsevier. You are right. We update this figure in V2. Major Issues The numbers in the paper do not seem to correspond directly to those in the dataset provided The data we report in the paper correspond with the filtered data, and they are coherent with the chosen criteria. It appears the article does not use DOAJ as the criterion for gold but the is_oa_journal field from unpaywall. This explains the variance between my analysis and that in the article for \"gold\" as defined here (16% vs 19% using the data provided, vs 21.5% given in Figure 1) Explained in previous points: Unpaywall includes 100% OA journals, DOAJ indexed or not. This issue is clearer stated in V2 of the paper. In Figure 5-7 I think there may be an error in the counting of OA articles, counting all those articles for which there is an 'is oa' entry and not only those where it is set to True. For the analysis is needed to exclude the articles not analysed by Unpaywall (and thus, have an empty OADOI field). If you do this, the numbers are ok. Thank you so much for your great review." } ] }, { "id": "65637", "date": "08 Jul 2020", "name": "Jonathon Alexis Coates", "expertise": [ "Reviewer Expertise Metaresearch", "preprints" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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: Arrizabalaga et al. address the important issue of accessibility in biomedical publishing. Utilising data from Unpaywall the authors investigate the open access status of COVID-19 articles published within the first four months of 2020. The majority of the COVID-19 literature investigated in this study are classified as bronze open-access, potentially subject to removal behind a paywall at any time. There is also a comparison to other epidemics (SARS-Cov-1 and MERS) and more recent literature that enables some comparisons between the literature.\nThere are inherent weaknesses to such a study due to the time period chosen which, due to the nature of the temporary open-access of many articles, is subject to change in the future. However, this is acknowledged by the authors in the text and can be further addressed through additional discussion. Overall, this is an important topic assessing the early phase of the pandemic with this study requiring some relatively minor changes.\n\nMajor concerns:\nClearer definitions for the different levels of open-access and licences, perhaps as a table. For those not familiar with the open-access terminology this would make the manuscript much clearer and easier to follow.\n\nBetter distinguish between open-access articles and those that are temporarily open-access through further discussion and analysis. The publisher motivations are highly important, particularly if a large proportion of the current bronze open-access subset is likely to be placed behind a paywall in the future.\n\nClear details on how data were collected, for example, was the data collected via the Unpaywall API or by a list of DOI's? This is particularly relevant for the date of collection, which will impact the results should others attempt to replicate as the authors themselves state in the limitations.\n\nFig. 1A is misleading, presenting all articles and the open access articles summed together. Data should be presented as a stacked bar not summing the articles with the open-access subset or as a Venn diagram. Moreover, Fig. 1C is confusing as currently displayed and may be better removed, with the information communicated in the text instead.\n\nIt would be nice to see the data for licences used for SARS-CoV-1, MERS, low grade glioma and peptic ulcers in the relevant figures. This is important information that helps to further understand the re-usability of open-access articles.\n\nMinor concerns:\n\nThe number of preprints has increased dramatically as a means of sharing COVID-19 research. It may be useful for the authors to discuss this especially considering the limited nature of some of the open-access COVID-19 literature.\n\nLicence “CC” should be “CC0” in text and figures throughout.\n\nClearer discussion over what the authors recommend as good open access principles (including the licence types).\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5770", "date": "12 Aug 2020", "name": "Olatz Arrizabalaga", "role": "Author Response", "response": "Dear Jonathon, Find here below, in red all the explanations to your helpful comments and insights. Arrizabalaga et al. address the important issue of accessibility in biomedical publishing. Utilising data from Unpaywall the authors investigate the open access status of COVID-19 articles published within the first four months of 2020. The majority of the COVID-19 literature investigated in this study are classified as bronze open-access, potentially subject to removal behind a paywall at any time. There is also a comparison to other epidemics (SARS-Cov-1 and MERS) and more recent literature that enables some comparisons between the literature. There are inherent weaknesses to such a study due to the time period chosen which, due to the nature of the temporary open-access of many articles, is subject to change in the future. However, this is acknowledged by the authors in the text and can be further addressed through additional discussion. Overall, this is an important topic assessing the early phase of the pandemic with this study requiring some relatively minor changes. Thank you very much for your thoughtful overall evaluation. We do agree with all of your comments and we are addressing all them and recommendations in Version 2 of the paper. Major concerns: Clearer definitions for the different levels of open-access and licences, perhaps as a table. For those not familiar with the open-access terminology this would make the manuscript much clearer and easier to follow. The authors agree completely with this perception. In the V2 of the paper we update these conclusions and added tables to some figures in order to follow easier the paper. Better distinguish between open-access articles and those that are temporarily open-access through further discussion and analysis. The publisher motivations are highly important, particularly if a large proportion of the current bronze open-access subset is likely to be placed behind a paywall in the future. Totally agree, we have included more results and conclusions about this in the new version. Clear details on how data were collected, for example, was the data collected via the Unpaywall API or by a list of DOI's? This is particularly relevant for the date of collection, which will impact the results should others attempt to replicate as the authors themselves state in the limitations. Yes you are right. Together with the new analysis we have updated the methodology section in order to clarify this issue. Fig. 1A is misleading, presenting all articles and the open access articles summed together. Data should be presented as a stacked bar not summing the articles with the open-access subset or as a Venn diagram. Moreover, Fig. 1C is confusing as currently displayed and may be better removed, with the information communicated in the text instead. Changed in version 2. It would be nice to see the data for licences used for SARS-CoV-1, MERS, low grade glioma and peptic ulcers in the relevant figures. This is important information that helps to further understand the re-usability of open-access articles. Yes, you are right. We have included this information in the version too. Minor concerns: The number of preprints has increased dramatically as a means of sharing COVID-19 research. It may be useful for the authors to discuss this especially considering the limited nature of some of the open-access COVID-19 literature. Yes you are right. Although we mention it in some of the sections of the article, perhaps it would be a good idea to be able to make a deeper analysis just about it since it might be a topic that gives for a whole paper. Licence “CC” should be “CC0” in text and figures throughout. Yes. It is CC0, It is update in the v2 or the paper. Clearer discussion over what the authors recommend as good open access principles (including the licence types). Hope what is in the new version conforms this point. Thank you so much for your great comments." } ] }, { "id": "65638", "date": "04 Aug 2020", "name": "Pilar Rico-Castro", "expertise": [ "Reviewer Expertise R&D policy making", "Open Access", "Open Science", "research infrastructures", "open repositories", "peer reviewed journals", "public policies" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 addresses how the recent COVID-19 pandemic has boosted open access practices among publishers and researchers, and it concludes that current practices include neither proper nor adequate licensing of research articles by publishers. Despite the fact that publishers comply with OA compromises with authors, they do not meet with larger open science requirements – especially those regarding scientific contents’ reusability. This paper opens up a very necessary discussion about the role that publishers can play as enablers or avoiders for making scientific knowledge findable, accessible, reusable, and interoperable, even when they fulfill formal open access requirements. The analysis is made for the early months of the COVID-19 pandemic, a time in history in which humans have been confronted with a vital need of scientific responses for their simplest every day routines. This extreme circumstance is used to light the real dimension of our dependence from open science, not only as researchers but as human beings, and to point each actor’s responsibility in providing the conditions for scientific advances to meet with the FAIR and the OS requirements.\nHowever, the work has a few weaknesses that need to be properly addressed.\nMajor concerns:\n1.- Need for clarification of the article’s objective and the research question. Under the “Introduction” section a variety of ideas are mixed and the research objective is not clearly stated. Mentions to the “scientific collaboration” with no previous context nor ulterior analysis, and sentences like “we wonder if the scientific community is ready to share and consume openly such information” contribute to blur the research objective. A clear statement on the research objective is needed.\n2.- Need for clarification of the research object. It is not clear whether or not pre-prints are included within the scope of the analysis. The paper needs an explicit declaration on that.\n3.- The comparative method has not been adopted correctly for the following reasons:\n- The comparability of the search “2019-nCoV OR 2019nCoV OR COVID-19 OR SARS-CoV-2 OR (Wuhan AND corona-virus)”, for which only articles published in a four months period has been considered, with the search “SARS CoV” OR “Severe Acute Respiratory Syndrome Coronavirus”, for which a three years period has been considered (2003 to 2006) and with the search “MERS CoV” OR “Middle East Respiratory Syndrome Coronavirus” for which a different three years period has been considered (2013 to 2016) needs a previous normalization. The time periods are very different (4 months vs. 3 years), and that invalidates the comparison. For sorting this out, the author could either normalize the data for all the periods analyzed to a \"month unit\", or use in their analysis only the first 4 months of all the health crises in comparison.\n- The authors are comparing an open period (this crisis is not yet over and we do not know how long it would last) with two closed crises. This should be acknowledged in the text as a methodological limitation.\n- The health crises under comparison contain large differences amongst them that require to be taken into consideration and to be acknowledged in the text as a methodological limitation. That the three situations have been classified as health emergencies is not enough for them to be comparable. They hold important differences regarding infection rates and death rates. The rapid spread of the recent pandemic has led governments all around the world to adopt never seen before very drastic measures (like lockdown) with a formidable impact on our economic system. Under this circumstance, a huge pressure has been put on the scientific community; therefore it has affected publication rates. In addition, the recent pandemic is taking place where the public debate about open access to scientific research is at its peak time. Many governments and funding agencies all around the world are launching OA policies (PlanS, as an example) and negotiating transformative agreements with large commercial publishers. All these conditions have a strong potential to affect OA availability of publications, both regarding publishers’ editorial practices and researchers’ publication patterns, thus affecting the comparison levels of the different periods considered in the analysis. All these elements should be acknowledged as difficulties for the comparison in the paper.\n4.- Unpaywall categories are not mutually-exclusive. This should be properly addressed and explained in the analysis. A publication can be Gold and Green OA simultaneously, and it can also be Hybrid and Green OA simultaneously. Moreover, Bronze category can be combined with each of the remaining three categories (Green, Gold, and Hybrid) as well as with the Gold-Green and Hybrid-Green combinations. The only ones that are mutually-exclusive are Gold and Hybrid categories. This opens a major methodological concern: whether data have been double counted or not. Therefore:\n- What compatibilities exist between the different categories should be properly explained.\n- A clarification about whether or not there is double counting needs to be made.\n- In the case that double counting has been avoided, authors must explain from which category the items have been removed from, and under which criterion.\n- Authors do not explain how they found out that Green and Hybrid papers are classified under Bronze category. This explanation should be included under the results section.\n5.- Clarify the role of CC licenses within the OS requirements. The relationship between CC licenses and OS reuse requirements is not properly mentioned in the text. Brief explanations to clarify what CC licenses are and what role they play is needed.\n6.- Delete non-evidence based conclusions. The following sentences are not based in any proven evidence or data:\n- “From the data, it can be seen that the number of articles published during the selected period increases daily”. There are no data referring to daily publications in the paper.\n- “Shortening of acceptance rates by journals is giving rise to information overload both for the scientific community but also for society, making it difficult to ascertain what really has a significant scientific value and as a consequence may affect decision-making”. This cannot be inferred from the analyzed data. Nothing has been proven about the shortening of acceptance rates by journals or about the scientific value of the publications. None of these issues have been addressed in the paper.\n- “In addition to the massive scientific production, after the pandemic declaration, publishers have made, not only COVID-19 but also previous SARS CoV-1 and MERS CoV related papers, openly available”. This cannot be inferred from the analyzed data and it has not been proven. (Actually, in my opinion, the most likely explanation for finding SARS CoV-1 and MERS CoV related papers in OA is that the embargo period has already expired.)\n- “… as the majority of the documentation is not free all the time, the number of subscriptions might be affected since it is possible that new non-subscribed readers obtained during this pandemic period have read articles from these journals and want to continue doing it.” This cannot be inferred from the analyzed data and it has not been proven. Actually, it is quite unlikely since scientific journals’ subscriptions are not decided nor negotiated by researchers, but by academic libraries.\n- “What is most interesting about the effect of the COVID-19 emergency on scientific research disclosure is what it says about the current publication model: it fails when a critical need arises for fast data dissemination”. This sentence from the conclusion section goes against the evidence presented in the analysis since authors have shown that of a total of 5,611 published articles related to COVID-19 pandemic, 4,986 were in OA in some way or another. Also, nothing has been proven about the speed of dissemination; therefore no conclusions can be drawn about this issue.\n- “We finally conclude that it seems clear that all stakeholders agree that Science only works when knowledge is shared.” There is no evidence to sustain this sentence. It should be either proven or deleted.\n7.- Strength evidence-based conclusions:\n- “While Gold OA makes papers available immediately by the publishing journal itself, the predominant Bronze OA category, found by the present study, means that papers are freely hosted…” This whole paragraph contains the main evidence-based conclusion of the work. The idea that OA is not enough, and that despite the fact that publishers put a multitude of works in open access in response to a certain situation (in this case pandemic) it that does not guarantee an open, findable, accessible, interoperable and reusable science, should be a strength in the paper.\n- “Our analysis demonstrates that the current alternative that is in use falls short of expectations of being the best model, since this fast opening lacks basic OA principles, which are required in order to be transparent, reusable and…” This sentence contains the second main evidence-based conclusion of the work. It should be a strength in the conclusions section of the paper.\nMinor concerns:\n1.- Need for brief definitions:\n- Definitions of Open Access and Open Science concepts as well as proper citations about both concepts are missing. Open Science means much more than Open Access. A proper brief definition of both concepts is needed for the reader not to mix them up.\n- “In order to analyse publications concerning COVID-19 and their level of openness, we have chosen PubMed instead of other multidisciplinary databases, like Web of Science (WoS) or Scopus”. Clarify in this sentence that PubMed, WoS, and Scopus are databases for bibliographic references.\n2.- Need for cites.\n- “In this unique context of the pandemic, publishers are announcing massive OA changes, primarily by making their corona-virus-related articles freely available through databases, such as PubMed Central, together with other public repositories”. This paragraph lacks proper citations and a more detailed explanation on the cited new practices launched by publishers that differentiates pre-print repositories from opening peer reviewed published articles.\n3.- Need for web references of Scopus, PubMed, MEDLINE, and PubMed Central (PMC), as it has been done for WoS.\n4.- Correct the expression “five categories” because there are only four (Gold, Hybrid, Green, and Bronze).\n5.- Clarification of the meaning of “Q1” in Figures 1, 2, 3, and 4. It is confusing since the reader tends to think of the 1st quartile of the JIF.\n\n6.- Change Figure 1a since it is confusing. It is not straightforward to see that the top portion is a part of the bottom portion. It looks like the addition of both is the total. There are more appropriate figures to show both the total and its proportion in a more intuitive manner.\n7.- Clarify Figure 1c. Figure 1c needs further clarification in the text about the meaning of \"Via page says license\", and \"Via free article\" categories.\n8.- Mention why the publishers’ and journals’ analysis has not been made for SARS CoV-1 nor MERS CoV searches. The analysis conducted for the three periods is different. No description regarding neither publishers nor journals has been made for publications about SARS CoV-1 nor MERS CoV.\n9.- Completing data in Figure 3a. The percentages in graph 3a add up no more than 68.6%. This means that there are 31.4% of the publications that are not included in the graph. This is important to be noticed since the remaining 31.4% is a higher figure than the largest category represented (29.7%). It is recommended to include a category \"others\" with 31.4% of the publishers. The dispersion of the data is very large. Focusing the analysis only on Elsevier, Wiley and Springer is reducing it to 54% of the data. This should be mentioned it in the text.\n10.- Figure 3b refers exclusively to 12.7% of the data. This should be mentioned it in the text.\n11.- Explain graph 4b in the text.\n12.- Change graph 4d. This graph is not very accurate. I suggest using a similar graph than the previous one (4c).\nFinally, this paper opens the door for further research to be done in the future, like the analysis of the relationship between the four categories of OA (Gold, Hybrid, Green, and Bronze), the CC licenses that they use, and the publishing practices of the different large publishing companies. It would be fantastic if the authors continue their work in this way.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5985", "date": "30 Sep 2020", "name": "Olatz Arrizabalaga", "role": "Author Response", "response": "Dear Pilar, Thank you very much for your comments. As the publication of the version 2 of the paper coincided with your review submission, some of your comments may not be implemented in this last version, but the vast majority are included in V2. Some your comments included in V2: - Strength evidence-based conclusions - All the minor concerns Please find here below, the explanations to some of your helpful comments and insights. - Need for clarification of the research object. It is not clear whether or not pre-prints are included within the scope of the analysis. The paper needs an explicit declaration on that. No pre-prints were included in the analysis. Mentioned in V2. - The comparability of the search “2019-nCoV OR 2019nCoV OR COVID-19 OR SARS-CoV-2 OR (Wuhan AND corona-virus)”, for which only articles published in a four months period has been considered, with the search “SARS CoV” OR “Severe Acute Respiratory Syndrome Coronavirus”, for which a three years period has been considered (2003 to 2006) and with the search “MERS CoV” OR “Middle East Respiratory Syndrome Coronavirus” for which a different three years period has been considered (2013 to 2016) needs a previous normalization. The time periods are very different (4 months vs. 3 years), and that invalidates the comparison. For sorting this out, the author could either normalize the data for all the periods analyzed to a \"month unit\", or use in their analysis only the first 4 months of all the health crises in comparison. That is a good point. Yes, we are aware of this and we mention it at limitations section of the second version of the paper. But also we find it interesting to see the huge difference when comparing only four months vs a first year from the outbreak of the other epidemics. Also, it is not possible to do a “monthly” analysis as many of the SARS and MERS papers lack a proper acceptance date. -The health crises under comparison contain large differences amongst them that require to be taken into consideration and to be acknowledged in the text as a methodological limitation. That the three situations have been classified as health emergencies is not enough for them to be comparable. They hold important differences regarding infection rates and death rates. The rapid spread of the recent pandemic has led governments all around the world to adopt never seen before very drastic measures (like lockdown) with a formidable impact on our economic system. Under this circumstance, a huge pressure has been put on the scientific community; therefore it has affected publication rates. In addition, the recent pandemic is taking place where the public debate about open access to scientific research is at its peak time. Many governments and funding agencies all around the world are launching OA policies (PlanS, as an example) and negotiating transformative agreements with large commercial publishers. All these conditions have a strong potential to affect OA availability of publications, both regarding publishers’ editorial practices and researchers’ publication patterns, thus affecting the comparison levels of the different periods considered in the analysis. All these elements should be acknowledged as difficulties for the comparison in the paper. This is also a good point. In the second version of the article we mention in the introduction section the reason for comparing with SARS 1 and MERS epidemics: it is because of the similar epidemiology based on the respiratory transmission (unlike Ebola or Zika epidemics) as well as the alarm generated during the first outbreak in the three cases. Regarding the OA scenario, it is true that it differs between the three situations, but this could also be a fact that the opening should be greater than the one we are seeing - Clarify the role of CC licenses within the OS requirements. Yes, you are right. We have updated figures in V2 in which this relationship is explained. - Delete non-evidence based conclusions. We have modified some these conclusions so that they don’t seem so convincing. Hope what is in the new version complies your comments. Thank you so much for your great ideas." } ] } ]	1	https://f1000research.com/articles/9-649
https://f1000research.com/articles/9-972/v1	11 Aug 20	{ "type": "Case Report", "title": "Case Report: Longitudinal assessment of a COVID-19 patient in the midst of a pandemic", "authors": [ "Rashid A. Chotani", "Syed S. Ashraf", "Fatima Aziz", "Shakeel M. Thakurdas", "Afham Chotani", "Alize Ashraf", "Khurram Nasir", "M. Rizwan Sohail", "Faisal H. Cheema", "Rashid A. Chotani", "Syed S. Ashraf", "Fatima Aziz", "Shakeel M. Thakurdas", "Afham Chotani", "Alize Ashraf", "Khurram Nasir", "M. Rizwan Sohail" ], "abstract": "Directional clinical evaluation and management of coronavirus disease (Covid-19) was initially presumptive based on the Wuhan data set as reported by World Health Organization (WHO). The current recommendations emanate primarily from the Chinese experience and subsequent Centers for Disease Control and Prevention (CDC) guidelines. Here we report a case with an “atypical” patient risk profile and variant longitudinal disease progression contrasting from existing recommendations. Our case report suggests that a universal 14-day quarantine timeline may not be sufficient; that correlation is needed between viral presence as determined by RT-PCR and a patient’s humoral response tested by serologic immunoassay of IgM & IgG. Hence, a clinical decision-making algorithm that can help clinicians clear a patient from “active infection” status would require testing that is sufficiently reliable, and should include serological testing for immunity.", "keywords": [ "COVID-19", "Coronavirus", "Pandemic", "Pneumonia", "SARS", "Respiratory", "Throat swab" ], "content": "Background\n\nCoronavirus disease 2019 (COVID-19) is a highly contagious viral pandemic caused by a novel strain of severe acute respiratory syndrome (SARS)-related coronavirus (CoV) named SARS-CoV-21. The virus is part of the family of coronaviruses which have been involved in previous infectious outbreaks, namely SARS 2003 and Middle East respiratory syndrome (MERS) 20122. Initially detected in December 2019 in the Hubei Province of China3, information related to its epidemiology and virulence is constantly evolving as the disease spreads across the world. Overall global prevalence of the disease is difficult to assess with reasonable accuracy due to a paucity of standardized global testing and reporting4, which in turn makes it difficult to accurately assess the mortality rate associated with the disease.\n\nAs of June 17, 2020, confirmed cases of COVID-19 are close to 8,200,000 with more than 444,000 deaths reported worldwide. The United States has become the epicenter of this pandemic with more than 2,137,000 confirmed cases and over 116,000 reported deaths5. The information related to Covid-19 is evolving on a daily basis and recent reports indicate that there may be other Covid-19 strains affecting different areas of the globe6.\n\n\nObjective\n\nTo describe the longitudinal assessment of a 50-year-old female, RT-PCR confirmed - COVID-19 patient, during an evolving pandemic and changing guidelines.\n\n\nClinical vignette\n\nWe present the case report of a 50-year-old South Asian female who came to our clinic in the middle of April 2020, complaining of intermittent fever ranging between 100-102°F, chills, night sweats, fatigue, and myalgia for the past six days. The patient reported no past medical history, no recent international travel history or exposure to any known COVID-19 positive patient and is a nutritionist by profession. At the clinic (day 6 of symptom onset, see Figure 1), she was noted to have a fever of 100.2℉. Two days prior (day 4 of symptoms, Figure 1), she reported testing negative for Streptococcus (group A strep) and influenza at an urgent care facility. During her visit at our clinic, her physical exam was unremarkable and her lungs were clear on auscultation. In view of her clinical presentation and negative strep and influenza test, we decided to test her for COVID-19 and an oropharyngeal swab was collected and sent for RT-PCR analysis. One day after her visit to our clinic (day 7 of symptoms, Figure 1), the patient developed shortness of breath (SOB) with persistent fever ranging between 100–102°F, chills, night sweats, fatigue and myalgia. She was started on a seven-day course of Levaquin (500 mg once daily) empirically for a presumptive diagnosis of community acquired pneumonia while awaiting RT-PCR results and albuterol inhaler (every four hours or as needed) for bronchospastic cough. The following day (day 8 of symptoms, Figure 1), she returned to the clinic, and during auscultation of the lungs, bibasilar crackles were noted, greater on her left lung compared to the right lung. She was then sent to the emergency department (ED) of a nearby hospital where a chest x-ray was performed on the same day (Figure 2). The chest x-ray revealed bilateral pulmonary infiltrates. Her laboratory work-up in the ED showed an elevated blood lactate level of 2.0mmol/L (normal: 0.5–1mmol/L), while her complete blood count and basic metabolic panel showed no abnormalities. Oxygen saturation rate was 97% and she was subsequently discharged to home. Three days after her visit to the ED (day 11 of symptoms, Figure 1), the patient still reported persistent SOB and was sent by our clinic to the ED for follow-up evaluation and possible need for chest CT given concerns for Covid-19 related viral pneumonia. At the ED, another chest x-ray was done which showed patchy opacities with air bronchograms in the left mid-lung and left lower lobe as well as similar patchy peripheral opacities at the right upper lobe, suggesting infectious etiology and worsening pneumonia (Figure 3). Oxygen saturation rate was still 97%, no other laboratory tests were reportedly done during this visit and she was again discharged to home. Four days later (day 15 of symptoms, Figure 1), the patient became afebrile (97.6℉) and reported not having any chills but still complained of significant fatigue and myalgia; her SOB, however, was improving. The next day (day 16 of symptoms, Figure 1), the patient returned to our clinic with a fever of 99.1℉. While her breathing was much better, she stated that she felt weak and had significant diffuse muscle pain. At the clinic, we performed a rapid COVID-19 specific IgM/IgG test, provided by Boston Biopharma, which came up positive for both IgM and IgG 16 days after the initial onset of symptoms (Figure 1). Routine labs were also done that showed anemia with hemoglobin 10.5g/dl (normal: 11.7–15.5g/dl) and hematocrit 33.1% (normal: 35–45%) with mean corpuscular volume 74fl (normal: 80-100fl), mean corpuscular hemoglobin 23.5pg (normal: 27–33pg), mean corpuscular hemoglobin concentration 31.7g/dl (normal: 32–36g/dl), platelet count 857,000/ml (140–400K/ml), an elevated C-reactive protein level (CRP) of 30.4mg/l (normal: <8.0), and creatine phosphokinase (CPK) of 39u/L (normal: 29–143 u/L). Her comprehensive metabolic panel (CMP), and white blood cell count with differential showed no abnormalities. On day 17 since symptom onset, the patient was afebrile (97.5℉) without dyspnea and no further labs were done after this point since the patient started to improve symptomatically. She did however complain of persistent weakness and myalgia. The patient’s husband and son had both remained asymptomatic during the entire episode and both tested negative with Boston Biopharma’s rapid COVID-19 antibody test on the same day as the patient was tested for antibodies (day 16 of patient’s symptoms, Figure 1). However, results from nasopharyngeal swabs taken from both the husband and son for RT-PCR analysis returned a week later were positive for the husband only. The husband’s repeat IgM and IgG, checked twice seven days apart, remain negative and he has continued to remain asymptomatic.\n\nA graphic representation of some of the relevant events with respect to the day of symptom onset shown as D1 (red bar on the timeline). Even though pneumonia is not a symptom we thought it would be helpful to easily visualize the onset of signs of pneumonia on the timeline. At no point was the patient hospitalized perhaps because the O2 saturation remained at or above 97%. SF, San Francisco; Pt, patient; GpA Strep, Group A Streptococcus; Influ, influenza; OP, oropharyngeal; CXR, chest x-ray; SOB, shortness of breath; Ab, antibody; CBC, complete blood count.\n\nThe patient returned to the clinic one day after the onset of shortness of breath and was referred to the emergency department of a nearby hospital where this x-ray was done. The x-ray shows patchy bilateral opacities, left greater than right, concerning for pneumonia.\n\nThe x-ray shows patchy opacities with air bronchograms in the left mid-lung and left lower lobe as well as similar patchy peripheral opacities at the right upper lobe suspicious for infectious etiology which appears mildly worsened.\n\nAll three individuals, the patient, her husband, and her son were interviewed again for any recollection of recent travel or secondary exposure. Her husband had returned just two weeks before the onset of her symptoms, after a week-long stay in San Francisco. Four days after his return from San Francisco, he travelled again to Atlanta for a day trip and attended a conference in Washington, DC before returning home the next day. Just a couple of days after the husband’s return the patient recalled going to an axe-throwing party with him and a group of friends. She also claimed that her husband developed a slight fever and a sore throat the day after this party and his symptoms lasted for at least three days (see Figure 1). Of note, the patient herself had developed a sore throat and low-grade fever by this time and this was the onset of all her reported symptoms. We also learned that four people from the group that attended the axe-throwing party reported “some” illness. One couple (Couple 1) had high fever and cough that lasted for 2–3 days and were told to self-quarantine for 14 days by their primary care provider (no information is available if they were tested). Couple 2 had been in San Francisco the same week as patient’s husband, but they did not meet there. This couple also reported being sick with fever and cough that lasted three days around the same time as her husband’s symptoms. They were also advised by their primary care physician to self-quarantine for 14 days. All cases from the axe-throwing party (husband, Couple 1 and Couple 2) recovered without any worsening of symptoms in 2–3 days, and prior to the patient’s development of her symptoms.\n\n\nDiscussion\n\nThe most common symptoms of Covid-19 are fever, cough, loss of energy or exhaustion, and to a varying degree, other bodily manifestations7. The symptoms and findings suggestive of disease progression that should raise concern are chest discomfort (pressure, tightness)8, confusion or change in mental status not otherwise related to another etiology9, and hypoxia without necessarily experiencing shortness of breath10. In more severe cases, infection can cause pneumonia, severe acute respiratory distress syndrome (ARDS), and even death. It is believed that ARDS and similar catastrophic respiratory changes may be related to a cytokine storm phenomenon11. The period within which the symptoms seem to manifest is wide, ranging from two to 14 days12. In our patient, a 50-year-old female with no international travel history, no co-morbidities but with potential exposure to multiple people who had traveled to a high Covid-19 exposure area, initial symptoms (fever, sore throat, chills, myalgia), appeared on the third day after being at the axe throwing party (a potential exposure event) and progressed in little over a week to dyspnea and worsening bilateral pneumonia. The husband, Couple 1 and Couple 2 seemed to have developed mild symptoms just a day or two after the axe-throwing party, suggesting that one of the members at this party was the source and probably an asymptomatic carrier. It is important to note that our patient who was Covid-19 positive (RT-PCR), developed bilateral pneumonia and was treated only with Levaquin and albuterol and recovered, indicating that in the absence of some major event such as a cytokine storm or ARDS, viral pneumonia due to SARS CoV-2 can clear by itself.\n\nIt is well established that the initial defense to a foreign antigen involves a complex immune response, both of innate and adaptive types. Antibody response in the form of IgM provides a critical defense during the early stages of a viral infection. Subsequent development of a longer-term IgG antibodies further augments the adaptive immune response and may eventually represent some form of immunological memory13. In our patient, both IgM and IgG were positive 16 days after symptom onset. Testing of Covid-19 IgM and IgG antibodies is therefore an effective method for confirming whether an individual has had the Covid-19 infection. This has value both in diagnosis and monitoring for early or late infectivity response. While viral replication usually wanes within a week or two without significant illness in most healthy individuals, antibody response, especially IgG antibody, typically rises for a few weeks after exposure and can remain elevated for long periods of time, sometimes even years, which is in fact the driving principle behind vaccination. For this reason, antibody testing will likely be the most valuable tool to assess the prevalence of SARS-CoV-2 infection and the level of exposure within the population14.\n\nA recent study published in the Lancet concluded that RT-PCR alone may not be sufficient in diagnosing a patient with Covid-19 in the lower respiratory tract or in assessing infectivity after few weeks of exposure15. Another study raises concern for the high false negative rates associated with nasopharyngeal and oropharyngeal swab sampling in an out-patient setting16. In view of these findings, it is our opinion that rapid antibody testing is an important tool for the clinical assessment and management of Covid-19. Such a test would be ideal in the third week from symptom onset based on our current knowledge of the immune response to viral pathogens. Subsequent RT-PCR is warranted if IgM is found to be positive so that the patient may be fully assessed for continued viral infectiousness and possible isolation. Additionally, given the asymptomatic status of both husband and son in such proximity to a Covid-19 patient, a rapid antibody test can be valuable for determining a person’s Covid-19 disease status and for surveillance17. In this case, the patient’s spouse tested positive with nasopharyngeal RT-PCR but negative with repeated serologic tests raising the possibility that he may be a nasal carrier.\n\nOur patient also had elevated CPK levels with persistent weakness and myalgia suggestive of post-viral myopathy. A broader survey of COVID-19 patients can inform if this is a significant sequela of the disease.\n\n\nConclusion\n\nThis case report helps to highlight the urgent need to answer some of the most pertinent questions associated with this pandemic such as the duration of quarantine, role of serologic antibody tests in diagnosing, management, and immunity assessment, and the role of various laboratory measurements in clinical follow-up. We recognize the limitations of this being a single case report (and of associated family members) and acknowledge that additional studies are necessary to determine how best to screen RT-PCR positive patients to assure that they are no longer infectious to their immediate contacts as well as the healthcare workers who are providing care for them18.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient. Written informed consent was also obtained from the mentioned relatives of the patient for publication of their clinical details.", "appendix": "Acknowledgement\n\nWe thank Dr. Sadiya Naseem, DNP, FNP-BC for following up on the patient and helping collect information\n\n\nReferences\n\nCoronaviridae Study Group of the International Committee on Taxonomy of Viruses: The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol. 2020; 5(4): 536–544. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeeri NC, Shrestha N, Rahman MS, et al.: The SARS, MERS and novel coronavirus (COVID-19) epidemics, the newest and biggest global health threats: what lessons have we learned? Int J Epidemiol. 2020; dyaa033. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang C, Wang Y, Li X, et al.: Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020; 395(10223): 497–506. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaffajee RL, Mello MM: Thinking Globally, Acting Locally — The U.S. Response to Covid-19. N Engl J Med. 2020; 382(22): e75. PubMed Abstract \| Publisher Full Text\n\nJohns Hopkins Coronavirus Resource. Reference Source\n\nWeise E: 8 strains of the coronavirus are circling the globe. Here’s what clues they’re giving scientists. Yahoo! news. 2020. Reference Source\n\nWang D, Hu B, Hu C, et al.: Clinical Characteristics of 138 Hospitalized Patients with 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China. JAMA. 2020; 323(11): 1061–1069. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInciardi RM, Lupi L, Zaccone G, et al.: Cardiac Involvement in a Patient with Coronavirus Disease 2019 (COVID-19). JAMA Cardiol. 2020; 5(7): 1–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFilatov A, Sharma P, Hindi F, et al.: Neurological Complications of Coronavirus Disease (COVID-19): Encephalopathy. Cureus. 2020; 12(3): e7352. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReport of the WHO-China Joint Mission on Coronavirus Disease 2019 (COVID-19). 2020. Reference Source\n\nMehta P, McAuley DF, Brown M, et al.: COVID-19: consider cytokine storm syndromes and immunosuppression. Lancet. 2020; 395(10229): 1033–1034. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaul G, Auwaerter MD: Johns Hopkins - Point of Care IT Guides. 2020. Reference Source\n\nWarrington R, Watson W, Kim HL, et al.: An introduction to immunology and immunopathology. Allergy Asthma Clin Immunol. 2011; 7 Suppl 1(Suppl 1): S1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhao J, Yuan Q, Wang H, et al.: Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019. Clin Infect Dis. 2020; ciaa344. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPan Y, Zhang D, Yang P, et al.: Viral load of SARS-CoV-2 in clinical samples. Lancet Infect Dis. 2020; 20(4): 411–412. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang W, Xu Y, Gao R, et al.: Detection of SARS-CoV-2 in Different Types of Clinical Specimens. JAMA. 2020; 323(18): 1843–1844. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuo L, Ren L, Yang S, et al.: Profiling Early Humoral Response to Diagnose Novel Coronavirus Disease (COVID-19). Clin Infect Dis. 2020; 71(15): 778–785. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLan L, Xu D, Ye G, et al.: Positive RT-PCR Test Results in Patients Recovered from COVID-19. JAMA. 2020; 323(15): 1502–1503. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "78496", "date": "15 Feb 2021", "name": "Ya-Dong Gao", "expertise": [ "Reviewer Expertise Allergy." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis Case Report described a laboratory-confirmed COVID-19 patient in detail, with longitudinal and dynamic assessment. As the authors stated, several implications can be drawn from it. Firstly, false-negative results of virus nucleic acid testing can occur. In our previous retrospective study (Zhang et al., 20201), 14.1% of finally laboratory-confirmed COVID-19 patients had a negative initial RT-PCR test. This may result from various factors, such as human errors when following the diagnostic kit protocol, poor sensitivity of reagents, the site and method of specimen sampling and collection times, low viral load in the sampled area during the early phase, and so on. In addition, serological measurement of IgM and IgG antibodies against the virus can be a useful and complementary tool (Li et al., 20202). The patient in the report was tested both positive for IgM and IgG 16 days after the onset of symptoms, but the RT-PCR result at this time was not clarified in the text. Moreover, asymptomatic individuals can be infectious, and they were likely to get infected in a party. Therefore, social distancing and personal protective measures, e.g., wearing a mask, are important.\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": "140766", "date": "14 Jun 2022", "name": "José López Castro", "expertise": [ "Reviewer Expertise Epidemiology of heart failure in elderly population: risk factors", "quality life health related and survival. Another line of research includes the study of prognostic biomarkers in COVID19 and genetics changes. Apart form this", "we are developing a study about advance care planning financed by the Spanish Government." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 clinical case of COVID19 that highlights the natural evolution of the disease and the possibility of diagnostic doubts, especially in the first days. It is well written but some issues need to be corrected:\nThe introduction should be shortened and the amount of data provided, which are fairly well known, should be reduced.\n\nThe inflammatory potential of the disease should be explained and that this can make the symptomatology longer and more aggressive (2 bibliographic references are attached for this).1,2\n\nThe bibliography should be reviewed and updated.\n\nExplain the different methods available for diagnosis of COVID19 and that the fact that an RT-PCR is positive after 14 days does not indicate that the disease is contagious.\n\nAfter these modifications, I believe it can be indexed.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-972
https://f1000research.com/articles/9-963/v1	10 Aug 20	{ "type": "Brief Report", "title": "Fast, easy and early (larval) identification of transparent mutant zebrafish using standard fluorescence microscopy", "authors": [ "Ralf Wenz", "Emily Conibear", "Laurence Bugeon", "Maggie Dallman", "Ralf Wenz", "Emily Conibear", "Laurence Bugeon" ], "abstract": "The availability of transparent zebrafish mutants (either TraNac: trab6/b6; nacw2/w2 or casper: roya9/a9; nacw2/w2) for live imaging studies together with the ease of generating transgenic lines are two of the strengths of the zebrafish model organism. The fact that transparent casper (roya9/a9;nacw2/w2) and silver nacre (nacw2/w2) mutants are indistinguishable by eye at early stages (1-5 days post-fertilization; dpf) means many fish must be raised and later culled if they are not transparent. To identify translucent mutants early and easily at the early larval stage (≤5 dpf) before they are classified as protected animals, we developed a simple screening method using standard fluorescence microscopy. We estimate that this procedure could annually save 60,000 animals worldwide.", "keywords": [ "tra", "nac", "trab6/b6nacw2/w2", "casper", "Zebrafish", "transparent", "translucent", "screening", "iridophore" ], "content": "\n\nEarly identification of TraNac I casper mutations in zebrafish larvae (5 dpf)\n\nEarly screening of zebrafish larvae could result in 60,000 fewer adult fish being raised and culled, annually worldwide. For each zebrafish mutant line, an approximate 75% reduction in animal use could be achieved.\n\nFast, early and easy identification of transparent zebrafish larvae at 5 dpf.\n\nReducing the number of animals raised by 75% concomitantly decreases the costs associated with animal husbandry\n\nScreening TraNac / casper mutants in zebrafish larvae\n\nAutomated screening of TraNac / casper mutants\n\n\nIntroduction\n\nThe zebrafish is a very popular vertebrate model organism, being the second most commonly used animal species in Great Britain. Of the 1.72 million procedures in 2018 purely relating to the creation and breeding of genetically altered animals, 223,600 (13%) were zebrafish (Home Office, 2019). This is because, amongst other beneficial features, one zebrafish female can produce several hundred eggs in a single clutch (Lawrence, 2011). Moreover, zebrafish lend themsleves to live imaging even at later stages of development due to the availability of transparent mutants. These transparent mutants are homozygous compound mutants known as TraNac (trab6/b6;nacw2/w2), and casper (roya9/a9;nacw2/w2) (Figure 1).\n\nThe pattern characteristic of Zebrafish colouration depends on three pigment cell types: melanophores, xanthophores and iridophores (Singh & Nüsslein-Volhard, 2015). Mutational inactivation of two of those chromatophores gives rise to transparent zebrafish (TraNac and casper). TraNac = trab6/b6; nacw2/w2 compound zebrafish mutants, nac = nacre, roy = roy orbison, tra = transparent, WT = wild-type.\n\nThere are two mutations involved in changing the pigmentation of zebrafish. The first is nacre (nacw2/w2). Nacre mutants do not have a functional transcription factor encoded by mitfa and therefore lack melanophores (Lister et al., 1999). This results in a uniformly silvery coloured ‘nacre’ zebrafish. The second mutation involved in pigmentation is roy orbison (roya9/a9), or roy hereafter. Roy has the identical frameshift and premature stop codon as the mutation transparent (trab6/b6), which will be referred to as tra (D’Agati et al., 2017). Both roy mutants (Ren et al., 2002) and tra mutants (Krauss et al., 2013) have an aberrant mitochondrial inner membrane protein 17 (Mpv17 protein) and therefore lack iridophores (D’Agati et al., 2017). This results in zebrafish that have no silver pigment but instead black spotted melanocytes. If both mutations, nac and roy / tra, are present and homozygous, the fish will lack melanophores and iridophores and are thus transparent (Figure 1).\n\nWhen one requires transparent TraNac or casper zebrafish to also express a specific transgene, the transgenic line of interest - commonly created on a wild-type (WT) background – is crossed with the transparent mutant line. The first generation will have a WT pigmentation phenotype. The incrossed second generation will be a mix of WT, silver nacw2/w2, spotted trab6/b6, and transparent mutants (TraNac or casper) in a ratio of 9:3:3:1 (Figure 2). While it is possible to identify WT and tra zebrafish before six days post fertilization (dpf) by simply screening for melanocyte pigmentation, it is currently not possible to distinguish between nacre and transparent TraNac / casper zebrafish before 6 dpf. Therefore all transparent looking 5 dpf fish (nacre and TraNac / casper) are currently raised to an age at which they can be distinguised, which is about 2 months post-fertilisation. At this point, not needed nacre fish can be culled. This means that even after removing all pigmented embryos before 5 dpf, ~75 % of the remaining second generation still must be culled at a later date. Therefore, a method that could identify transparency before 6 dpf; the stage at which they become protected animals under the Animals (Scientific Procedures) Act, 1986 would potentially reduce the number of protected animals culled every year by thousands.\n\nThe earliest possible transparent phenotype after a wild-type and TraNac fish have been bred (P generation) is the second generation (F2). However, at that point, only 6.25% of all fish will theoretically be transparent, due to the genetic inheritance pattern of both tra and nacre alleles being passed on in their mutated form (trab6 & nacrew2). As indicated in the Punnet square, there are 16 possible combinations of genes (T indicating functional tra allele and t represents mutated trab6, while N indicates functional nacre and n mutated nacrew2). The 16 possible combinations can result in 4 different phenotypes WT pattern, tra pigmented pattern, silvery nacre, transparent TraNac with the associated ratio of 9:3:3:1. nac = nacre, tra = transparent, WT = wild-type.\n\nWe have identified a way to screen for TraNac and casper mutants at early stages using conventional stereo-microscopy. This new approach has two major advantages: firstly, this approach allows the early and easy identification of transparent zebrafish for experiments; and secondly, crossing WT zebrafish onto transparent backgrounds will not require any culling of unwanted intermediate nacre fish of the second generation at a legally protected age. Therefore, this approach could save 60,000 adult fish worldwide every year (the detailed analysis of this metric follows in the dicussion).\n\n\nMethods\n\nZebrafish were maintained using standard practices and all procedures conformed to the Animals (Scientific Procedures) Act, 1986 of Government of the United Kingdom as well as the Directive 2010/63/EU of the European Parliament. Animals were maintained under UK Home Office project licence number P5D71E9B0. All efforts were made to minimize animal suffering by daily surveillance of animal health and water conditions, enriching the environment using live feed, by not performing invasive procedures that may in any way harm the animal and by reducing the number of animals necessary.\n\nRearing and maintenance of the WT, TraNac, nacre and casper fish was carried out at 28.5°C on a 14 h light/10 h dark cycle. AB fish strain of both sexes were used and were sourced locally from Imperial College Central Biological Services. The system water was derived from deinoised water reconstituted with sodium chloride salt to a final conductivity of 750 μS ± 50, while pH levels were kept within boundaries of 7.0 ± 0.2. Fish were housed in 3-litre see-through polycarbonate tanks of the Aquatic Habitats Z-Hab System (MBKI, Nottingham, UK) with a density of around 5–7 fish per litre. Feeding of fish was done according to stages twice a day, once in the morning and once in the evening: 6 dpf – 8 dpf fish were fed with ZM000 by ZM systems, 9 dpf – 14 dpf fish were fed with ZM100 (ZM systems), 15 dpf – 2 months post-fertilization old fish were fed with ZM200 (ZM systems), while any older adults were fed with pellet food by Hikari Tropical. As part of the environmental enrichment, adult fish were fed live Artemia salina once a day in the morning.\n\nThree experiments with two experimental groups each were done. We compared the correct identification of TraNac vs nacre fish, as they are indistinguishable by eye at 5 dpf. In the TraNac groups of the three separate experiments were 16, 9, and 12 fish, respectively; while in the nacre groups of the three separate experiments were 19, 13 and 15 fish, respectively. We had, using power calculations, determined that 15 adult fish per group would render 90% power at a 0.05 significance level, a standard deviation of 2, and a difference in mean of 2.5. In this study, we obtained on average 14 fish per group. This, however, still rendered a 88% power and which still is accepted as scientifically valid according to the documentation of the NC3Rs’ Experimental Design Assistant (NC3Rs EDA, 2020).\n\nZebrafish from 0 days post-fertilization (dpf) to 5 dpf were reared in Petri dishes in system water with with 3x10-5% methylene blue. For anesthesia, fish were transferred into a new Petri dish containing 4.2% (168 µg/mL) MS-222 (Sigma, E10521-50G) in system water with 3x10-5% methylene blue. Fish were screened by observing different fluorescent patterns of the eyes as illustrated in Figure 3 and Figure 4. A Leica M205 FCA stereomicroscope using a Leica DFC7000 T camera, the Leica LAS X software, and the Leica EL6000 external light source for fluorescence excitation was used for all experiments. The filters used were the Leica ET mCherry (Article Number: 10450195; Excitation nm: ET560/40x; Emission nm: ET630/75m) as well as the ET GFP (Article Number: 10447408; Excitation nm: ET470/40x; Emission nm: ET525/50m). Once screened according to phenotype, fish were transferred to a new Petri dish containing only system water and methylene blue. The screening procedure takes, depending on practice, approximately 5-10 minutes, per dish of 100 fish.\n\nUsing different channels (brightfield, GFP and mCherry), different patterns of colouration in the zebrafish eye become apparent between TraNac zebrafish larvae (I), in contrast to both nac (E), and WT (C) larvae. Although both fluorescent channels, GFP and mCherry appear to be equally useful for screening for eye pigmentation, by experience, the red fluorescent mCherry channel is easier for distinguishing in practice. nac = nacre, roy = roy orbison, tra = transparent, WT = wild-type.\n\nFish are anaesthetized in 4.2% MS-222 (168 µg/mL). Thereafter simple visual screening of larvae allows WT and tra fish to be discarded. The next step is fluorescent microscopy screening using the mCherry filter for different colour patterns in the eyes of the fish (see Figure 3). If TraNac or casper fish are desired, one screens for fish without any eye pigmentation. TraNac = trab6/b6; nacw2/w2 zebrafish mutants.\n\nTo determine the screening efficiency of the above procedure (see also Figure 3 and Figure 4), the screened 5 dpf fish were allowed to develop to the adult stage, the stage at which skin pigmentation can be clearly seen (Figure 1). If adult fish had silver pigments in their skin they were identified as nacre fish, and if they had neither silver pigments nor melanocytes in their skin they were identified as TraNac fish. Fish were recorded as correctly screened at 5 dpf if the identified 5 dpf phenotype matched the phenotype at the adult stage.\n\n\nResults\n\nWe showed that TraNac and casper fish do not have autofluorescence in their eyes, when subject to fluorescence microscopy in the mCherry channel, in contrast to WT fish. Through this finding we were able to develop a simple two-step process to identify transparent TraNac or casper zebrafish as outlined in Figure 4. First, after anaesthetising the fish, embryos that were observed by eye to have black pigments were discarded. These were either WT or tra mutants that still produce melanophores. Subsequently, using a fluorescent stereo-microscope with an mCherry filter, fish that did not have visible red eyes (see Figure 3, I) were identified. Those fish with visible red eyes using the mCherry filter were nacre mutants and would develop iridophores in the future (Figure 3, C & F). Of note, while iridophores are already present at 3 dpf in the eyes of zebrafish (Gur et al., 2018), screening at 5 dpf was found to be easier.\n\nUsing this screening procedure, we were able to correctly identify ~99% of zebrafish embryos at 5 dpf, either nacrew2/w2 or TraNac (Table 1). In three separate screening experiments (n = 84 fish) only one fish was wrongly identified at 5 dpf. This was confirmed by observing their pigmentation pattern at the adult stage. Similarly, casper zebrafish, which carry the same mutations as TraNac fish (D’Agati et al., 2017) can be screened with the same methodology. This screening method allows the identification of fully transparent zebrafish mutants before 6 dpf, the age at which they become protected animals under the Animals (Scientific Procedures) Act, 1986.\n\nIn three separate experiments, fish from three separate clutches were screened for either TraNac or nacre phenotype. As a result, six screens for either TraNac or nacre zebrafish were performed. Screening for the desired zebrafish phenotype was done at 5 days post-fertilization and successful identification was assessed ≥ 2 months post fertilization.\n\nTraNac = trab6/b6; nacw2/w2 zebrafish mutants.\n\n\nDiscussion\n\nThe method presented herein could lead to many thousands of animals not being culled after the age at which they become legally protected animals under the Animals (Scientific Procedures) Act, 1986. We estimate that every two years around 120,000 fish worldwide could be saved. This is based on two approximations: (1) We carried out a literature review which identified about 3% of labs using a mutation that is involved in making TraNac / casper zebrafish. We searched the online database Scopus, for articles published in the year 2018 with the following keywords and Boolean operators: zebrafish OR danio rerio AND adult. To obtain a managable number of papers we further narrowed down the search to only return papers in the subject area of ‘Immunology and Microbiology’. Of the 527 papers we could access 509. We found that about 2.9% (15 papers) used zebrafish with a roy, tra or nacre mutation. (2) A recent estimate by the NC3Rs states that there are about 3250 institutions in the world that use zebrafish (Lidster et al., 2017).\n\nTaking the two approximations together with common husbandry practices, we can therefore make a reasonable estimate about the number of fish that are culled unneccessarily every year. If about 3% of all 3250 institutions use TraNac / casper zebrafish, that means that there are ~100 institutions that keep these fish. In our lab we keep 15 transgenic lines on a transparent background, but in the following we will assume most labs only keep 10. On average per transgenic line we keep 40 fish. To establish one tank with 40 TraNac or casper zebrafish about 120 non-transparent nacre fish would be culled (see Figure 2 for Punnet square and resultant ratio of 9:3:3:1). This means that in one lab alone, to establish 10 transgenic lines of transparent fish, 1,200 fish would be culled. Since there are roughly 100 institutions that keep transparent zebrafish, the total number of fish that would be culled is about 120,000. Further, since it is common practice to outcross the lines every 2 years onto a WT background to enrich genetic diversity, 120,000 fish that would need to be culled are generated every two years for breeding purposes alone.\n\nIt is likely that a large fraction of these 120,000 fish could be saved in the future, because the uptake of this method is simple and the barriers are so low. The microsopy is easy, fast, and inexpensive. In fact, by implementing this method significant long term cost savings are likely, as 75% less fish need to be raised to adulthood. Besides these practical benefits, this approach also has several scientific benefits. It is now possible to identify TraNac / casper mutants early in development, allowing one to study the downstream impact of these mutations while having siblings from the same parental clutch, which would have previously been impossible.\n\nIn conclusion, the method presented allows for fast, early and easy identification of transparent (TraNac and casper) zebrafish and could lead to 60,000 adult fish being saved every year worldwide.\n\n\nData availability\n\nOriginal microscopy image files from Figure 3 are provided in a TIF format. To view these files, they should be imported into an appropriate image processing program such as FIJI (Schindelin et al., 2012).\n\nZenodo: Fluorescent microscopy images of larval zebrafish of either TraNac, Nacre or WT background. http://www.doi.org/10.5281/zenodo.3813755 (Wenz, 2020)\n\nThis project contains the following underlying data:\n\n- Nacre_5dpf.tif\n\n- TraNac_5dpf.tif\n\n- WT_5dpf.tif\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe would like to thank the Central Biological Services at Imperial College London for their support of our work with animals and the care for thereof. Moreover, we would like to thank Dorottya Pólos for providing, on occasion, fish used in the study.\n\n\nReferences\n\nD’Agati G, Beltre R, Sessa A, et al.: A Defect in the Mitochondrial Protein Mpv17 Underlies the Transparent Casper Zebrafish. Dev Biol. 2017; 430(1): 11–17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGur D, Nicolas JD, Brumfeld V, et al.: The Dual Functional Reflecting Iris of the Zebrafish. Adv Sci (Weinh). 2018; 5(8): 1800338. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHome Office: Annual Statistics of Scientific Procedures on Living Animals Great Britain 2018. 2019. Reference Source\n\nKrauss J, Astrinides P, Astrinides P, et al.: Transparent, a Gene Affecting Stripe Formation in Zebrafish, Encodes the Mitochondrial Protein Mpv17 That Is Required for Iridophore Survival. Biol Open. 2013; 2(7): 703–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLawrence C: Chapter 23 – Advances in Zebrafish Husbandry and Management. Methods Cell Biol. 2011; 104: 429–51. PubMed Abstract \| Publisher Full Text\n\nLidster K, Readman GD, Prescott MJ, et al.: International Survey on the Use and Welfare of Zebrafish Danio Rerio in Research. J Fish Biol. 2017; 90(5): 1891–1905. PubMed Abstract \| Publisher Full Text\n\nLister JA, Robertson CP, Lepage T, et al.: Nacre Encodes a Zebrafish Microphthalmia-Related Protein That Regulates Neural-Crest-Derived Pigment Cell Fate. Development. 1999; 126(17): 3757–67. PubMed Abstract\n\nNC3Rs EDA: NC3Rs EDA. 2020. Reference Source\n\nParliament of the United Kingdom: Animals (Scientific Procedures) Act 1986. 1986. Reference Source\n\nRen JQ, McCarthy WR, Zhang H, et al.: Behavioral Visual Responses of Wild-Type and Hypopigmented Zebrafish. Vision Res. 2002; 42(3): 293–99. PubMed Abstract \| Publisher Full Text\n\nSchindelin J, Arganda-Carreras I, Frise E, et al.: Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012; 9(7): 676–682. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSingh AP, Nüsslein-Volhard C: Zebrafish Stripes as a Model for Vertebrate Colour Pattern Formation. Curr Biol. 2015; 25(2): R81–92. PubMed Abstract \| Publisher Full Text\n\nWenz R: Fluorescent microscopy images of larval zebrafish of either TraNac, Nacre or WT background. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3813755" }	[ { "id": "70416", "date": "02 Sep 2020", "name": "Paul C. Evans", "expertise": [ "Reviewer Expertise Referee suggested by the NC3Rs for their scientific expertise and experience in assessing 3Rs impact." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFigure 3. There is autofluorescence from the abdomen. Is this the yolk sac? Please label this.\n\nIs there a complication with fluorescent transgenic embryos? I imagine that most lines will not have altered fluorescent eyes but perhaps this can be commented on.\n\nAre the 3Rs implications of the work described accurately? 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? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "69153", "date": "04 Sep 2020", "name": "Robert Hindges", "expertise": [ "Reviewer Expertise Zebrafish models", "imaging", "neuroscience", "development", "axon guidance", "visual system", "synapse", "cell adhesion 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 article, Wenz and colleagues present a simple and straight-forward method to screen zebrafish larvae at early stages, in order to identify different pigmentation mutant genotypes. The screening method is based on autofluorescence signal in the eyes of the larvae, visible in the red channel. This therefore abolishes the need to keep larvae longer than 5 days post fertilisation and raise them until adult stages in order to sort out the required genotype.\nOverall, I think this is an excellent approach, which will be helpful for the scientific community using these pigmentation mutants for their imaging experiments.\n\nI have some minor comments:\n\nLegend to Figure 1: as the genotypes are explained for TraNac I would suggest to also include the genotype description for casper.\n\nFigure 3 has no scale bars\n\nPage 4, last paragraph before Methods: Typo “discussion”\n\nPage 5, last line of first paragraph: missing space between “animaland”\n\nLegend to Figure 4: the statement to “screen for fish without any eye pigmentation” is misleading, as these larvae have still pigmented eyes (in contrast for example to the Crystal mutant (roy, nacre, alb mutant), not mentioned here). It should rather say: “screen for fish without autofluorescence signal in the eyes.\n\nPossible limitation of this screening method for transgenic lines having red-fluorescent reporters expressed in the eye could be discussed.\n\nAlthough I appreciate the motivation of the authors to give an estimated number of saved adult fish per year, there are a lot of assumptions made. In addition, only a particular subject (Immunology & Microbiology) is used. I think it would be helpful to know the concrete number of adult fish saved in the authors’ laboratory over the last year in addition to the more speculative numbers based on Scopus searches.\n\nAre the 3Rs implications of the work described accurately? 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? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-963
https://f1000research.com/articles/8-1390/v1	08 Aug 19	{ "type": "Systematic Review", "title": "Efficacy of mitomycin-C on anterior urethral stricture after internal urethrotomy: A systematic review and meta-analysis", "authors": [ "Gampo Alam Irdam", "Irfan Wahyudi", "Andy Andy", "Gampo Alam Irdam", "Irfan Wahyudi" ], "abstract": "Background: Mitomycin-C is an agent that plays an important role in the tissue healing process and scar formation. This study aims to investigate the efficacy of mitomycin-C in treating anterior urethral stricture following internal urethrotomy. Methods: Studies evaluating the efficacy of mitomycin-c for anterior urethral stricture post urethrotomy were searched using PubMed, Scopus, ScienceDirect, EBSCOHost, Cochrane Reviews, and OVID as directory databases. Terms used in the searching process were “mitomycin-c” or “mitomycin”, “urethral stricture”, “urethral stenosis”, “internal urethrotomy”, “optical urethrotomy” and its synonyms. Every randomized controlled trial study conducted in human subjects was included. Study appraisals were conducted in accordance with Oxford University Center for Evidence-Based Medicine guidelines. The conclusion of each study was summarized and the calculation of fixed effect from every study was conducted in meta-analysis. Results: Included in this study were three studies involving 231 patients. All of them reported less recurrence of in patients treated with mitomycin-c post urethrotomy (p<0.001). The fixed risk ratio of all studies was 0.32 with 95% confidence interval (0.19-0.54). All studies also reported less stricture length after treatment with mitomycin-c, but there were not statistical differences between with or without treatment group. Conclusion: Mitomycin-C has efficacy in treating anterior urethral stricture after internal urethrotomy. However, the inclusion of relatively few studies may affect the strength of this review and further studies need to be done.", "keywords": [ "mitomycin-c", "urethral stricture", "internal urethrotomy" ], "content": "Background\n\nUrethral stricture often impairs quality of life and may result in a large economic burden1. There are several procedures available for treating this condition, ranging from minimally invasive procedures like internal optical urethrotomy (IOU) to invasive procedure such as urethroplasty, with or without grafting, and tissue engineering2. However, despite the methods available, urethral stricture often recurs. Several manipulations have been tried to prevent urethral stricture, such as indwelling catheter insertion, urethral calibration procedure, and home self-catheterization. Unfortunately, repeated instrumentation can cause scar formation. Moreover, it can also complicate subsequent reconstruction, which can lead to several complications3,4. On the other hand, there have been several studies evaluating the effects of antifibrotic drugs such as glucocorticoid and mitomycin-C on urethral strictures. Mitomycin-C is an agent that has the potential to inhibit mitosis, fibroblast proliferation, formation of blood vessels, and synthesis of protein and collagen. This agent plays role in tissue healing process and scar formation by reducing the release of matrix proteins by inhibiting proliferative fibroblasts5.\n\nTo our knowledge, there have not been any systematic reviews or meta-analyses regarding the efficacy of mitomycin-C in treating anterior urethral stricture post internal urethrotomy. Thus, the present study aims to investigate the efficacy of mitomycin-C in treating anterior urethral stricture post internal urethrotomy. We hope that by conducting this review and analysis, a definite conclusion regarding the efficacy of such treatment could be achieved.\n\n\nMethods\n\nThis systematic review was conducted based on guidelines from the Oxford University Center for Evidence-Based Medicine6. Our present study aims to determine whether mitomycin-C provide better efficacy compared to controls (without mitomycin-C) in adult patients with anterior urethral stricture after internal urethrotomy.\n\nTo be considered for inclusion, the included studies were required to be randomized controlled trials (RCTs) study investigating efficacy of mitomycin-C as the additional treatment to internal urethrotomy in anterior urethral stricture. We expanded the searching by including related studies suggested by the databases. Year of publishing was not considered as inclusion criterion. Any study until September 22th 2018 was included. The primary outcome measures were efficacy of mitomycin-C administration, determined by risk ratio for proportion results and mean difference for continuous data. Animal studies, case series, case report, editorials, and book chapters were excluded.\n\nTo find suitable studies to be included in this review, we used PubMed, Scopus, ScienceDirect, EBSCOHost, Cochrane Reviews, and OVID as directory databases. We used combination of keywords “((((((mitomycin c[MeSH Terms]) OR mitomycin[MeSH Terms]) OR mitomycin c)) AND ((((((((((((((((urethral stricture[MeSH Terms]) OR urethral strictures[MeSH Terms]) OR stricture, urethral[MeSH Terms]) OR strictures, urethral[MeSH Terms]) OR urethral stenosis[MeSH Terms]) OR urethral stenoses[MeSH Terms]) OR stenosis, urethral[MeSH Terms]) OR stenoses, urethral[MeSH Terms]) OR urethral stricture) OR urethral strictures) OR stricture, urethral) OR strictures, urethral) OR urethral stenosis) OR urethral stenoses) OR stenosis, urethral) OR stenoses, urethral)) AND “urethra/surgery”[MeSH Terms]) AND Humans[Mesh]”. We also used term “human” as limiting term to exclude every study that was not conducted on human subjects.\n\nWe evaluated the study using appraisal worksheet for randomized clinical trial from Oxford University Center for Evidence-Based Medicine to stratify the risk of bias6. Using Revman 5.3 software, inputted data of stricture length (in mm) and recurrence number from all selected studies were analyzed. Data were analyzed using the homogeneity index (I2) and forest plots. Calculation of fixed effect was also done using Revman 5.3 to show relative risk/risk ratio for recurrence rate variable, mean difference for stricture length, dan p-value for both variables. We summarized the conclusion of each study at the table along with its appraisal.\n\n\nResults\n\nSearching process (searching strategy showed in Figure 1) by using six databases found 47 study articles. There were 28 articles eliminated after title and abstract screening. The remained 19 articles were reduced to six articles after eliminating duplicates, leaving six full text articles to be reviewed. Based on study design, we eliminated three articles, leaving three articles to be summarized in systematic review and meta-analysis.\n\nThree selected studies were conducted in 2007, 2015, and 20162,4,7. All studies evaluated the effectivity of mitomycin-C given after internal urethrotomy for anterior urethral stricture. From these selected articles, two evaluated the usage of submucosal injection of mitomycin-C for anterior urethral stricture after urethrotomy. How mitomycin-C was injected differed in both studies; one study used 0.1 mg mitomycin-C in 2 ml distilled water injected in four quadrants while another study used 0.1% mitomycin-C injected into three quadrants. One study evaluated the intraluminal injection of mitomycin-C in a hydrogel base. It consists of 0.8 mg mitomycin-C with 1 cm3 propylene glycol and water to PF-127 poloxamer. The hydrogel base was injected through a small feeding tube to reach the site of stricture. All studies applied mitomycin-C after internal urethrotomy procedure and were conducted in populations with different age means. Each of studies’ quality was assessed using guide from Oxford University Center for Evidence-Based Medicine; this is explained in Table 1.\n\nMMC, mitomycin C.\n\nLoE, level of evidence; RR relative risk; ARR, absolute risk reduction; RRR, relative risk reduction; NNT, number needed to treat.\n\nWe included the studies in which stricture was measured using retrograde urethrogram or ultrasonography of the urethra. The outcome that measured was recurrence rate (percentage) and stricture length (mm) after treatment.\n\nAll selected articles stated that mitomycin-C had a significant effect on preventing or delaying urethral stricture recurrence post internal urethrotomy. All studies reported that the time-based recurrence rates in the two groups differed, where lower recurrence rates were found in the group given mitomycin-C2,4,7. The forest plot in Figure 2 showed that recurrence rate are homogenous in all analyzed studies (I2 <0.001, p=0.55). This was performed by using fixed effect model in the forest plot. This forest plot suggests that there were significant differences between cases and control group. The mean recurrence rate was 0.32 (95%CI=0.19 to 0.54).\n\nTest for heterogeneity chi-square = 1.18; df(Q) = 2; p-value < 0.0001; I2 < 0.001.\n\nOn the other hand, in stricture length, all studies showed that there were not significant differences between the groups that received and did not receive mitomycin-C administration (p=0.38). Figure 3 showed homogenous studies in stricture length (I2 < 0,001; p=0.38) analysis. The mean difference was -0.02 (95%CI=-0.27 to 0.23).\n\nTest for heterogeneity chi-square = 0.23; df(Q) = 2; p-value = 0.89; I2 < 0.001.\n\n\nDiscussion\n\nUrethral stricture is a serious complication in male patients, which causes great morbidity and considerable health-related costs. This condition often results in voiding dysfunction, which will affect quality of life. Moreover, this voiding disfunction can trigger chain of events leading to renal failure. Majority of the recommended treatment methods have low success rate8. A recent survey revealed that 86% of American urologists prefer internal urethrotomy when treating anterior urethral stricture9. However, internal urethrotomy is associated with a high rate of urethral stricture recurrence, ranging from 20 to 60%10,11. Although several methods have been introduced to lower recurrence rate, including clean intermittent self-catheterization12, numerous authors has labeled this a failure as it could not fully prevent recurrence of urethral stricture.\n\nDespite unclear pathophysiology of urethral stricture, a pathophysiological mechanism suggested is fibrosis caused by excessive synthesis of collagen and altered extracellular matrix13,14. Therefore, any drug or procedure which can delay fibrosis after internal urethrotomy would lead to an increase of surgical success rates and patient comfort, and therefore decrease treatment costs. Therefore, many studies have been performed to explore different molecules the can prevent fibrosis and urethral stricture recurrence4,13–16.\n\nMitomycin-C is an alkylating antineoplastic antibiotic, produced by Streptomyces caespitosus. Mitomycin-C can inhibit DNA synthesis by linking adenine and guanine, resulting in DNA cross-linking. It can suppress cellular RNA and protein synthesis, and is not cell cycle specific8. Therefore, it can delay the healing process by preventing replication of fibroblasts and epithelial cells, as well as inhibiting collagen synthesis6,14. It has been shown that mitomycin-C can improve the success rates of trabeculotomy and myringotomy by preventing proliferation of fibroblasts and development of fibrosis18,19.\n\nThe anti-fibroblast activity mechanism of mitomycin-C is unknown. Experts have suggested that the reduction of fibroblast activity may be mediated by myofibroblasts apoptosis2,4,11. As the wound closes, apoptosis of myofibroblast and vascular cells increases, indicating that this is the mechanism by which granulation tissue will lead to scarring20,21.\n\nAll the studies included in this review treated the two groups equally and had relatively small loss-to-follow-up rates. A common problem with all the studies included in this review is that there was no clear blinding statement. In the study by Mazdak et al.4, it was not stated whether there was a randomization process in the study method. On the other hand, although Ali et al.2 had randomized its subjects, age characteristics in the two groups were significantly different.\n\nAll studies support the use of mitomycin-C to prevent or delay anterior urethral stricture after internal urethrotomy. This was confirmed by a lower rate of recurrence rate in patients treated with mitomycin-C patients2,4,7; we found that those who had mitomycin-C administered had lower incidence of recurrence during one year and 18 months of follow up (RR = 0.32, P < 0.001). This was also confirmed by a series of cases by Farrell et al.22, Farrell et al.23, and Sourial et al.24 Mazdak et al.11 injected mitomycin-C into the submucosal layer of the urethra and reported lower rates of stricture recurrence in patients with mitomycin-C injection. On the other hand, some researchers proposed that submucosal injection might lead to an increase in complication rates and a decrease in the duration of the effective dose within the tissue19. Ayyildiz et al.25 assessed the efficacy of mitomycin-C in preventing urethral scar by means of applying the agent topically to the traumatized region in rats. They concluded that mitomycin-C applied locally reduced fibrosis significantly in a dose-independent manner.\n\nIn stricture length, we found no statistically difference between the mitomycin-C-treated and control group in all studies. However, Mazdak et al. 4 and Moradi et al.7, even statistically showed no significant differences between these groups, the mitomycin-C-treated group showed a decrease in stricture length. The results of the study by Mazdak et al.4 were 0.76 mm (range:0.5-1 mm) in the mitomycin-C group compared to 0.84 mm (range:0.5-1 mm) in the control group after the procedure. Moradi et al.7 observed stricture lengths of 10.7±5.9 and 9.55±4.15 mm for the control and mitomycin-C groups, respectively.\n\nAlthough all studies support the use of mitomycin-C to prevent or delay post-urethrotomy urethral stricture, the results of this review need to be followed up with caution. The limitation of this study can be seen from only a few studies that discuss this topic. Some of the existing studies are not enough to be applied to a wider population, given that selected studies were carried out only in Iran and Pakistan (and thus may not be representative of different ethnic groups)2,4,7. Therefore, although the side effects reported in the studies reviewed are minimal, their application needs to be carried out wisely and cautiously. Research related to this in the future can still be done with different populations.\n\nDue to short period of follow up time in all studies, some authors2,4 concluded that the study of the use of mitomycin-C in this case needed firm results regarding long-term success. Mazdak et al.4 added that stricture may recur within two years after internal urethrotomy.\n\n\nConclusion\n\nMitomycin-C could be used as a potential additional treatment for anterior urethral strictures after internal urethrotomy. However, further studies are required to investigate the safety and efficacy of this method for treating anterior urethral strictures, as only a limited number of studies presently exist.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nOpen Science Framework: PRISMA checklist for ‘Efficacy of mitomycin-C on anterior urethral stricture after internal urethrotomy: A systematic review and meta-analysis’. https://doi.org/10.17605/OSF.IO/APU9B26.\n\nThe updated PRISMA checklist is available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nZaid UB, Lavien G, Peterson AC: Management of the Recurrent Male Urethral Stricture. Curr Urol Rep. 2016; 17(4): 33. PubMed Abstract \| Publisher Full Text\n\nAli L, Shahzad M, Orakzai N, et al.: Efficacy of mitomycin C in reducing recurrence of anterior urethral stricture after internal optical urethrotomy. Korean J Urol. 2015; 56(9): 650. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcCammon K, Zuckerman J, Jordan G: Surgery of the penis and urethra. In: Wein AJ, Kavoussi LR, Partin AW, eds. Campbell-Walsh Urology. 11th ed. Philadelphia: Elsevier; 2016; 938.\n\nMazdak H, Meshki I, Ghassami F: Effect of mitomycin C on anterior urethral stricture recurrence after internal urethrotomy. Eur Urol. 2007; 51(4): 1089–92. PubMed Abstract \| Publisher Full Text\n\nSu C, Sui T, Zhang X, et al.: Effect of topical application of mitomycin-C on wound healing in a postlaminectomy rat model: an experimental study. Eur J Pharmacol. 2012; 674(1): 7–12. PubMed Abstract \| Publisher Full Text\n\nCritical appraisal for randomized controlled trial. Center of Evidence-Based Medicine University of Oxford; 2019.\n\nMoradi M, Derakhshandeh K, Karimian B, et al.: Safety and efficacy of Intraurethral Mitomycin C Hydrogel for prevention of post-traumatic anterior urethral stricture recurrence after internal urethrotomy. J Inj Violence Res. 2016; 8(2): 75–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKurt O, Gevher F, Yazici CM, et al.: Effect of mitomycin - C and triamcinolone on preventing urethral strictures. Int Braz J Urol. 2017; 43(5): 939–45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFerguson GG, Bullock TL, Anderson RE, et al.: Minimally invasive methods for bulbar urethral strictures: a survey of members of the American Urological Association. Urology. 2011; 78(3): 701–6. PubMed Abstract \| Publisher Full Text\n\nDubey D: The current role of direct vision internal urethrotomy and self-catheterization for anterior urethral strictures. Indian J Urol. 2011; 27(3): 392–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSantucci R, Eisenberg L: Urethrotomy has a much lower success rate than previously reported. J Urol. 2010; 183(5): 1859–62. PubMed Abstract \| Publisher Full Text\n\nKhan S, Khan RA, Ullah A, et al.: Role of clean intermittent self catheterisation (CISC) in the prevention of recurrent urethral strictures after internal optical urethrotomy. J Ayub Med Coll Abbottabad. 2011; 23(2): 22–5. PubMed Abstract\n\nNagler A, Gofrit O, Ohana M, et al.: The effect of halofuginone, an inhibitor of collagen type I synthesis, on urethral stricture formation: In vivo and in vitro study in a rat model. J Urol. 2000; 164(5): 1776–80. PubMed Abstract \| Publisher Full Text\n\nMazdak H, Izadpanahi MH, Ghalamkari A, et al.: Internal urethrotomy and intraurethral submucosal injection of triamcinolone in short bulbar urethral strictures. Int Urol Nephrol. 2010; 42(3): 565–8. PubMed Abstract \| Publisher Full Text\n\nSciarra A, Salciccia S, Albanesi L, et al.: Use of cyclooxygenase-2 inhibitor for prevention of urethral strictures secondary to transurethral resection of the prostate. Urology. 2005; 66(6): 1218–22. PubMed Abstract \| Publisher Full Text\n\nGiovannini UM: Treatment of scars by steroid injections. In: Wound Repair Regen. 2002; 10(2): 116–7. PubMed Abstract \| Publisher Full Text\n\nBradner WT: Mitomycin C: a clinical update. Cancer Treat Rev. 2001; 27(1): 35–50. PubMed Abstract \| Publisher Full Text\n\nEstrem SA, Vanleeuwen RN: Use of Mitomycin C for maintaining myringotomy patency. Otolaryngol Head Neck Surg. 2000; 122(1): 8–10. PubMed Abstract \| Publisher Full Text\n\nFontana H, Nouri-Mahdavi K, Lumba J, et al.: Trabeculectomy with mitomycin C: outcomes and risk factors for failure in phakic open-angle glaucoma. Ophthalmology. 2006; 113(6): 930–6. PubMed Abstract \| Publisher Full Text\n\nDesmoulière A, Redard M, Darby I, et al.: Apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar. Am J Pathol. 1995; 146(1): 56–66. PubMed Abstract \| Free Full Text\n\nLee DA, Lee TC, Cortes AE, et al.: Effects of mithramycin, mitomycin, daunorubicin, and bleomycin on human subconjunctival fibroblast attachment and proliferation. Invest Ophthalmol Vis Sci. 1990; 31(10): 2136–44. PubMed Abstract\n\nFarrell MR, Lawrenz CW, Levine LA: Internal Urethrotomy With Intralesional Mitomycin C: An Effective Option for Endoscopic Management of Recurrent Bulbar and Bulbomembranous Urethral Strictures. Urology. 2017; 110: 223–7. PubMed Abstract \| Publisher Full Text\n\nFarrell MR, Sherer BA, Levine LA: Visual Internal Urethrotomy With Intralesional Mitomycin C and Short-term Clean Intermittent Catheterization for the Management of Recurrent Urethral Strictures and Bladder Neck Contractures. Urology. 2015; 85(6): 1494–9. PubMed Abstract \| Publisher Full Text\n\nSourial MW, Richard PO, Bettez M, et al.: Mitomycin-C and urethral dilatation: A safe, effective, and minimally invasive procedure for recurrent vesicourethral anastomotic stenoses. Urol Oncol. 2017; 35(12): 672.e15–672.e19. PubMed Abstract \| Publisher Full Text\n\nAyyildiz A, Nuhoglu B, Gülerkaya B, et al.: Effect of intraurethral Mitomycin-C on healing and fibrosis in rats with experimentally induced urethral stricture. Int J Urol. 2004; 11(12): 1122–6. PubMed Abstract \| Publisher Full Text\n\nAndy: PRISMA Checklist - Efficacy of mitomycin-C on anterior urethral stricture after internal urethrotomy: A systematic review and meta-analysis. 2019. Publisher Full Text" }	[ { "id": "59108", "date": "03 Feb 2020", "name": "Laetitia M. O. de Kort", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe subject of this review is interesting and relevant to clinical practice. The review is well written. I have some comments:\nThe literature search was until September 2018. Why is an update missing?\n\nComplications and side effects of the MMC injections were not mentioned.\n\nThere was no clear definition of recurrence of the stricture. As this is a major subject of dispute in urethral surgery, this issue should be addressed, at least in the discussion.\n\nThe length of the stricture was not mentioned.\n\nWere these all primary strictures? Should be addressed.\n\nThe first publication was in 2007. Yet, MMC injection is not implemented in clinical practice for prevention of urethral stricture. It should be discussed why this is the case.\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? Partly", "responses": [ { "c_id": "5388", "date": "03 Apr 2020", "name": "Andy Andy", "role": "Author Response", "response": "Thank you for your review. We have revised our publication according to your comments and we will upload it as soon as possible. But some comments I would like to answer them The literature search was until September 2018. Why is an update missing? We have searched with our search strategy and there are no updates until September 2018. The first publication was in 2007. Yet, MMC injection is not implemented in clinical practice for prevention of urethral stricture. It should be discussed why this is the case. It has been stated clearly that the evidence for MMC injections are not enough for it to be implemented in clinical practice. Therefore, we require further research. We are open to any suggestion for improving this study, thank you" } ] }, { "id": "59109", "date": "17 Feb 2020", "name": "Farhad Shokraneh", "expertise": [ "Reviewer Expertise Evidence Synthesis", "Systematic Review", "Information Retireval", "Automation of Systematic Reviews", "Living Evidence", "Scoping Review", "Cochrane Review", "Database", "Clinical Practice Guideline", "Overview", "Rapid Review", "Randomized Controlled Trial", "Open Data", "Medical Journalism", "Outcome", "Intervention", "Cochrane." ], "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 conducted the first systematic review to answer this research question. It is a valuable effort.\n\nAbstract\nEBSCOhost and Ovid are not databases. They are search interfaces or search engines for other databases. Please add the name of the databases that you search using these two tools.\n\nPlease add the exact search date (dd/mm/yyyy).\n\nPlease state the reason for not using Cochrane Risk of Bias tool for assessing risk of bias of RCTs.\n\nPlease add rationale for using fixed effect model.\n\nThe conclusion is so strong. With limited evidence, it is not easy to say that MMC has safety, efficacy, or long-term efficacy.\nMethods\nThe search date is older than a year (17 months old). Please update the search. I spent a few minutes and identified following recent studies:\n- Azzawi (2018)1\n- Islam et al. (2019)2\n\nEBSCOhost and Ovid are not databases. They are search interfaces or search engines for other databases. Please add the name of the databases that you search using these two tools.\n\nThe search strategy reported for PubMed needs serious attention by a search expert such as a librarian or information specialist and through using PRESS checklist for peer-reviewing search strategies. If authors have no access to such person they can post a task on taskexchange.cochrane.org and request a volunteer to help them in exchange to offering Acknowledgement or Authorship, depending on their contribution.\n\nWhen the number of results is little, usually the authors should seek other sources such as references of included studies, citations to relevant studies, contacting the authors of relevant studies, etc. Depending on amount of time and resources available for the team, I request them to use other methods to make sure no studies are missing.\n\nPlease structure Inclusion and Exclusion Criteria to subheadings: Patients, Intervention, Control/Comparison, and Primary and Secondary Outcomes.\n\nI wondered why side effects of interventions have not been listed as collected outcome data.\n\nPlease add separate headings for Screening, Data Extraction, Assessment of Risk of Bias, and explain their processes.\n\nPlease follow the PRISMA reporting guideline items one by one.\n\nPlease state the reason for not using Cochrane Risk of Bias tool for assessing risk of bias of RCTs while it is available within RevMan.\n\nPlease share your RevMan file as Appendix in Open Science Framework so that the readers could reproduce your analysis.\n\nPlease add rationale for using fixed effect model.\nResults\nPlease follow the original PRISMA flow diagram in which duplication is before screening.\n\nI2 is not the only way to notice heterogeneity. Actually, I2 may not work well with small sample size and low number of studies. The other ways is to look at the heterogeneity in study details such as characteristics of population (age – compare Age in Moradi with age in Mazdak –, sex, etc.), interventions (method of administration or dosage), and outcomes (methods of measurement). Also N/As in the table are missing details that you may get contacting the authors and they may contribute to heterogeneity. Based on what I see in your description, the method of administration is different among studies plus age of the patients varies across studies.\nDiscussion\nThe first four paragraph of the discussion are not discussing the results and they seem to be relevant to Background.\n\nImpact of small sample size, heterogeneity of included studies, adverse effects, subjectivity of outcome measure, not using placebo for control group on interpreting these results are some of the items that could have been discussed here. I also refer the authors to a commentary written by Ng and Chan on Mazdak et al. as the first RCT3.\nI thank the authors for listing some of the limitations this reviews. I think a new version of this review should be submitted, before being approved.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? No\n\nIs the statistical analysis and its interpretation appropriate? Partly\n\nAre the conclusions drawn adequately supported by the results presented in the review? No", "responses": [ { "c_id": "5456", "date": "03 Jun 2020", "name": "Andy Andy", "role": "Author Response", "response": "Thank you for the feedback, we have revised according to most of your comments. Update also has been implemented. As for contacting the authors for N/A in tables, we have tried contacting the authors but there is no answer until now." } ] } ]	1	https://f1000research.com/articles/8-1390
https://f1000research.com/articles/9-187/v1	13 Mar 20	{ "type": "Research Article", "title": "Evaluation of antioxidant, antibacterial and wound healing activities of Vitex pinnata", "authors": [ "Nurul Ashifah Shafie", "Nur Atiqah Suhaili", "Hussein Taha", "Norhayati Ahmad", "Nurul Ashifah Shafie", "Nur Atiqah Suhaili", "Hussein Taha" ], "abstract": "Background: Vitex pinnata is one of the many plants known for its ethnomedicinal properties. However, the pharmacological properties of this plant have not been well studied. This study aims to determine the antioxidant, antimicrobial and wound healing properties of the methanolic extract of the leaves and the hexane, chloroform and ethyl acetate fractions.\n\nMethods: The leaves of Vitex pinnata underwent methanol extraction and the methanol extract was fractionated with hexane, chloroform and ethyl acetate solvents. The antioxidant activity was determined using the DPPH radical scavenging assay. The antimicrobial activity was assessed by disc diffusion assay against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. For the wound healing studies, the methanolic extracts of V. pinnata were used to prepare ointments with compositions of 10% (w/w) and 50% (w/w), which were evaluated for wound healing activity in an excision wound model in Wistar rats. Results: All the extracts showed antioxidant activities, with the ethyl acetate extract having the highest DPPH radical scavenging activity, followed by the methanol, chloroform and hexane extracts. Similarly, their quercetin equivalent concentrations were 33.1, 31, 20.3 and 4.5 mg/mL, respectively. Except for the methanol extract, the disc diffusion assay showed that the extracts demonstrated species-specific antibacterial activities, with the ethyl acetate extract showing antibacterial activities against all four tested strains. The wound healing activity of the high dose treated group (50% [w/w]) shows significant increase of wound contraction when compared to the control group. Conclusion: In the current study, the ethyl acetate extract showed activity for all tested bacteria and also had the highest DPPH activity. The methanolic extracts of V. pinnata leaves show modest wound healing activity in an excision wound model.", "keywords": [ "Vitex pinnata", "antimicrobial", "antioxidant", "wound healing", "wound excision" ], "content": "Introduction\n\nVitex pinnata is a common ethnomedicinal plant locally known as ‘kulimpapa’ in Brunei Darussalam and East Malaysia. According to Goh et al. (2017), it is traditionally used as a treatment for stomach aches, fever, body aches and lesions. The bark may be used for abdominal pain, while the young shoots are utilized as sanitizing agents and deodorants. The roots and bark of this plant have been used in herbal baths and also taken orally in the form of decoction or herbal tea while the leaves may be eaten raw. Traditionally, the leaves were prepared into poultice and applied to wounds to induce fast healing (Burkill et al., 1966; Goh et al., 2017; Sahu & Barik, 2007; Suksamrarn & Sommechai, 1993).\n\nPlants have been known to be a potent source of medicinal compounds and anti-oxidants. Oxidative stress produced through a chain reaction by free radicals can be a contributing factor to the pathophysiology of various conditions including cardiovascular dysfunction, atherosclerosis, inflammation, carcinogenesis, reperfusion injury and neurodegenerative diseases (Aruoma, 1998). Due to the increasing safety concerns with the consumption of synthetic antioxidants, alternative sources of antioxidants from natural origins, especially from plants, are currently in demand (Stankovic et al., 2016).\n\nAlthough various antibacterial agents of synthetic origin are available, bacterial resistance to current antibacterial agents is growing (Andersson & Hughes, 2010). Furthermore, available antibiotics can also cause side effects. For this purpose, plants can be good sources of antimicrobial agents due to their secondary metabolites (Nascimento et al., 2000). Previous studies have reported the antifungal activity of V. pinnata (Ata et al., 2009), however its antibacterial and antioxidant activity has not yet been reported. In order to understand the possible effects of Vitex pinnata on wound healing, we have sought the use of animal models to reflect human wound healing conditions. Wistar rats were selected in our study because of their availability. In addition to this, the use of Wistar rats in this study provides a more mainstream model for understanding the wound healing process upon treatment with the extract. The use of this species and excision wound model also allows us to make a comparison of the wound healing process with other available literature involving a similar approach to ours. The scientific objective of this study is to determine the stage by stage process of wound healing such as general wound morphology, wound closure and presence of inflammatory cells, which could only be carried out in an animal model.\n\nIn this study, we report the antibacterial, antioxidant and wound healing activities of V. pinnata from Brunei Darussalam.\n\n\nMethods\n\nThe leaves of V. pinnata were collected at the Universiti Brunei Darussalam Botanical Research Centre (UBD BRC) in Brunei Darussalam. Species identification was kindly carried out by the botanist at the UBD BRC. The plant samples were shade dried for a few weeks and grinded prior to solvent extraction.\n\nA mass of 300 g of the ground leaves was exhaustively extracted with 1.5 L of methanol using Soxhlet extraction. The resulting methanol extract was then vacuum filtered and evaporated using a rotary evaporator. The methanol extract was further partitioned with different solvents in increasing order of polarity i.e. hexane, chloroform and ethyl acetate. A total of 10 g of the methanol extract was redissolved in approximately 1 L of methanol and 10 mL of distilled water, followed by the addition of 500 mL of hexane. After shaking vigorously, the mixture was left to stand to allow layers to form between the solvents. Once formed, the hexane layer was collected and the procedure was repeated with the other solvents (chloroform and ethyl acetate). Each solvent was evaporated with a rotary evaporator and subsequently air dried in a fume hood.\n\nThe percentage yield of extract was determined using the formula: Extraction yield (%) = [Dry weight of extract obtained / Dry weight of material used for extraction] × 100%. Prior to the antioxidant and antibacterial tests, each extract was re-dissolved in methanol at 1000 μg/mL and 500 mg/mL, respectively.\n\nThe antioxidant or radical scavenging activity (RSA) was determined by DPPH radical scavenging assay according to Awang-Jamil et al. (2019) with minor modifications. Each extract with a volume of 0.2 mL was mixed with 1 mL of 40 μg/mL of DPPH methanolic solution. For the control, 0.2 mL of methanol was used instead of the extract. After about 30 minutes, the RSA was determined by measuring the absorbance at 517 nm using a UV spectrophotometer. The assay was carried out in triplicate. The RSA (measured as the percentage of DPPH scavenged) was calculated using the formula: RSA (%) = [Acontrol – Asample]–/Acontrol × 100%, where Acontrol refers to the absorbance of the control and Asample refers to the absorbance of the extract.\n\nTo measure the quercetin equivalent (QE) concentration of each extract, the RSA of quercetin at six different concentrations (1, 5, 10, 20, 25 and 35 µg/mL) were also similarly measured. A standard calibration curve was prepared by plotting RSA (y-axis) against quercetin concentration (x-axis). Subsequently, the linear regression of the standard calibration curve (y = 2.74x + 2.77; R2 = 0.997) was employed for the estimation of QE concentration.\n\nThe microorganisms used in this study consisted of four different strains of bacteria: two Gram-positive bacteria, Staphylococcus aureus (ATCC-29213) and Bacillus subtilis (ATCC-11774); and two Gram-negative bacteria, Escherichia coli (ATCC-11775) and Pseudomonas aeruginosa (ATCC-27853). Each bacterial strain was grown in nutrient broth, and prior to the test, overnight bacterial culture was diluted to an absorbance of 0.08 to 0.1 (equivalent to 0.5 McFarland standard), measured with a UV spectrophotometer at 600 nm.\n\nAntibacterial activities of the extracts were determined by disc diffusion method according to Abdullah et al. (2019) with minor modifications. A volume of 10 µl of each extract was applied onto a filter paper disc (6 mm in diameter) and left to dry. Methanol was used as the negative control and streptomycin sulphate (antibiotic) as the positive control. Each standardised bacterial culture was then swabbed onto Mueller-Hinton agar (MHA) plates. The impregnated discs were then loaded onto the swabbed MHA plates. The zone of inhibition was recorded after incubating the plates at 37 °C for 24 hours. Each test was carried out with at least three replicates.\n\nEthical statement. All work involving animals was approved by the Universiti Brunei Darussalam University Research Ethics Committee [approval reference: UBD/FOS/E2(g)]. All animal procedures were performed in accordance with Universiti Brunei Darussalam guidelines on the care and use of animals for research and internationally accepted guidelines. All investigators declare that every effort was taken to ameliorate harm to the animals via close monitoring for signs of pain and distress, and attended to in case signs of pain and distress was observed in accordance with the Universiti Brunei Darussalam guideline on care and use of animals for research.\n\nAnimals. Male Wistar rats aged between 8–10 weeks and weight range between 150–200g were used for the wound healing study. The animals were obtained from and housed in the Universiti Brunei Darussalam animal facility. The rats were fed with a standard maintenance pellet diet (Cat # 1324, Altromin, Germany) and access to food and water was provided ad libitum. Animals were housed in cages with wood shavings as bedding and maintained under standard lab conditions (12/12h light dark cycle; 25°–30°C). Prior to carrying out the experimental procedures, animals were assessed for their general health, determined by their behavioral responses to the handler and other general parameters such as hands-on physical examination, condition of the animal’s body, physical and observable abnormalities. The welfare of the animals was attended to throughout the experiments such as by the provision of food and water, avoidance by the handler of conditions that cause suffering, pain and disease. Animals with obvious signs of pain and distress during the experiment would be excluded from the study.\n\nControl animals were placed between three to four animals per cage and following the wound excision procedure, animals were placed in individual cages throughout the duration of the experiment. Upon completion of the experiments, animals were euthanized using CO2 asphyxiation followed by cervical dislocation prior to tissue collections for histological analysis.\n\nFormulation of ointment. The methanolic leaf extracts of V. pinnata were formulated into ointments with pure petroletum jelly as the base. Herbal ointments were prepared in two doses, which is the low dose extract consisting of 10% (w/w) extract and high dose extract containing 50% (w/w) extract. The maximum concentration that was stable in the base was 50% (w/w).\n\nExcision wound. All procedures were refined in order to minimize any negative impact and pain to the animals resulting from the excision wound procedure. All procedures were carried out in the Universiti Brunei Darussalam animal facility at the same time of day between 10.00–13.00hrs. Prior to making the excision wound, all treated animals were anaesthetized using diethyl ether by inhalation anesthesia. Diethyl ether was chosen as the anesthetic because its action is also accompanied by analgesic properties. Animals were exposed to one drop of diethyl ether placed in a cotton wool and placed at one end of a conical tube. There was no close or direct contact between the cotton ball and the nose of the animal. Depth of anesthesia following exposure to diethyl ether was determined by responses to reflexes such as voluntary movements from stimuli such as extending of legs. Once deep anesthesia has been established, the animal excision wounds were performed as previously described (Umachigi et al., 2008). The dorsal fur of the animals was shaved and an area of about 100mm2 (10 × 10 mm) was excised using a sharp scalpel with disposable steel blades. Rats were then individually housed and the extract and ointment were applied topically on the wound area at the same time of day (between 10am –1pm) in the respective groups for a period of 21 days.\n\nAnimal grouping and dosing. Animals were randomly grouped into three groups, each group containing six rats. The numbers of animals (n) used in these experiments were based on previous studies in our laboratory and experiments of this nature (Nagar et al., 2016) and is also the minimum that is required to detect any significant difference between the treatment groups. Simple randomization was carried out when assigning the animals to groups in order to avoid any bias. Introduction of bias is reduced or eliminated as during the early stages of the experiment; i.e. during grouping, allocation of animals to treated and control groups was carried out randomly. Wound area determination was carried out by a validated instrument (Image J software, National Institute of Health, USA) which minimizes subjectivity of observer during assessment of wound areas.\n\nFollowing this, all animals within each group received the same treatment throughout the duration of the experiment. The rats of Group I were treated with pure petroletum jelly only (negative control). Group 2 and Group 3 were treated with ointments containing 10% (w/w) leaf extract (low dose) and 50% (w/w) leaf extract (high dose), respectively. The ointment was topically applied to the wounds on alternate days for a period of three weeks.\n\nWound area determination. Wound area was measured using Image J software (National Institute Health, USA) (Figure 1). The rate of wound contraction was measured as the percentage of reduction of wound size. The percentage of wound contractions were calculated as previously described (Shi et al., 2013): Wound contraction (%) = 100 × [(Original wound area – area on measured post-wounding day) / Original wound area]\n\nA) Anaesthetized rat with the scale placed near the wound site, labelled with name, post-wounding day and scale bar of 10 mm for measurement analysis. B) Calibration of Image J with known distance in image. C) The outline of the wound is drawn to measure wound area.\n\nHistological analysis. Skin tissue samples from the wound and its vicinity were taken for histopathological analysis at post wounding days 3, 7, 14 and 21. Sections at 10µm thickness were prepared and stained with hematoxylin and visualized.\n\nStatistical analyses were conducted with one-way ANOVA and a post hoc Tukey’s HSD test. These were performed using R 3.3.3 software. Values of p < 0.05 indicated statistical significance.\n\n\nResults and discussion\n\nStatistical analysis indicated that the RSA of the different extracts were significantly different to each other. The ethyl acetate extract had the highest RSA and QE concentration, indicating it was the most potent in inhibiting free radicals compared to the other extracts (Table 1). This was followed by the methanol extract, the chloroform extract and finally the hexane extract.\n\nThe values are shown as average ± standard deviation of three replicates.\n\nDifferent solvents used for extraction have been reported to extract different compounds (Basri et al., 2017). It has been previously reported that phenolic content increased with the increasing polarity of solvent (Barchan et al., 2014; Belyagoubi et al., 2016). The extracts from V. pinnata in the present study possibly contained different types of phenolic compounds with different antioxidant capacities. Zhang (2015) has studied the effects of solvents on the phytochemical contents. It was found that the extraction of total flavonoids significantly increased in polar solvents, which might contribute to the antioxidant activity in V. pinnata.\n\nIn the present study, the ethyl acetate and hexane extract showed the highest and lowest radical scavenging activities amongst the extracts, respectively. Similarly, a previous study conducted by Vats (2012) on cowpea (Vigna unguiculata) also showed that ethyl acetate extract had the best DPPH radical scavenging activity. Moreover, the highest phenolic content was also found in the ethyl acetate extract, implying that most phenols were soluble in ethyl acetate. The lowest scavenging activity was in the hexane extract, which could be attributed to its low amount of phenolic content.\n\nClosely related plant species have been shown to have similar phytochemical constituents (Diris et al., 2017). The antioxidant activities of V. pinnata’s close relatives, V. agnus-cactus, V. negundo and V. trifolia have also been determined using various solvents, and was found that the fruits and leaves of these Vitex plants were significantly capable of scavenging free radicals (Rashed, 2013; Saklani et al., 2017), signifying that Vitex plants are potential sources of promising antioxidants.\n\nDetected zones of inhibition as shown in Table 2 indicated the presence of antibacterial activities in the extracts of V. pinnata leaves at 500 mg/mL.\n\nThe values are shown as average ± standard deviation of at least three replicates. Negative control did not show any inhibition zone as expected whereas positive control (streptomycin sulphate) showed inhibition zones ranging from ~20 to 30 mm.\n\nThe results generally showed the presence of antibacterial activities in the hexane, chloroform and ethyl acetate extracts and no antibacterial activity detected in the methanol extract. As shown in Table 2, the ethyl acetate extract could inhibit all the bacterial strains as compared to chloroform extract, which could only inhibit a total of three bacterial strains, and hexane extract, which could inhibit two strains.\n\nAll animals were continuously observed and assessed for their general health status throughout the duration of the experiment. We did not observe any adverse effects in these animals such as infections of the excised region or any behavioral changes following the excision wounding and during the recovery period. The difference in wound area was observed from post-wounding day 7 in all experimental groups (Figure 2). The wound contraction at post-wounding day 7 was determined to be slightly higher for animals in the negative control group when compared to extract-treated groups (Figure 3). At post-wounding day 14, wound contraction in extract-treated groups were considerably higher when compared to the control group and particularly in animals receiving the higher concentration of extract (Table 3). Complete wound closure was observed in both the low dose 10% (w/w) and high dose 50% (w/w) extract treated groups at post-wounding day 21, indicating a faster rate of epithelialization compared to normal wound healing in the control group.\n\n denote significant changes of wound contraction at day 14 in comparison to control group, p < 0.01.\n\nValues are mean ± SEM; **p < 0.01 compared to control animals on similar treatment days. The starting experiment had n=6 animals per group. One animal was sacrificed for histological analysis at day 3, day 7 and day 14. There was n=3 animals remaining at day 21 which is the final day of the experiment.\n\nOur study demonstrated a dose dependent effect of the methanolic extract on wound healing, as evident from the wound closure observed for the 10% (w/w) vs 50% (w/w) extract treated animals. General wound morphology in post wounding day 3 shows a well-developed scab formation in all groups (Figure 2). We did not observe any adverse effects in both control and extract treated animals. At day 14, there was almost complete closure of the wound in the high dose group (Figure 2).\n\nWe observed a high density of inflammatory cells in the low dose and high dose treated tissues (Figure 4). The low dose and high dose treated animals showed epithelialization at day 7, whereas the control group showed epithelialization at day 14. Inflammatory cells become prominent, which may include neutrophils, followed by macrophages and mast cells that emigrate from nearby tissues, playing a crucial role in combating infection (Martin & Leibovich, 2005).\n\nDay 3 control and treated rats: presence of inflammatory cells is evident. Collagen deposition observed in the high dose treated animals at day 14 (x400).\n\nBased on histological observations, granulation tissue from all experimental groups displayed increased infiltration of fibroblast cells, indicative of active proliferation (Figure 4). Among all of the experimental groups, high-dose treated group showed greater wound contraction at post-wounding day 14 (Table 3). At post-wounding day 21, the wound in the high-dose treated group showed a more densely packed organization of collagen and connective tissues compared to the low dose-treated and control group.\n\nBased on the wound contraction evaluation and histological analysis of the wound, our study has demonstrated a modest wound healing effect in the V. pinnata high-dose treated group followed by low-dose treated group as compared to control group (Figure 2). Phytochemical analysis reports have revealed that the petroleum ether, ethyl acetate, methanol and aqueous leaf extracts of V. pinnata contain varying amounts of the alkaloid, anthocyanidins, aucubins, coumarins, flavonoids, flavanols, gallic tannins, iridoids, proteins, reducing compounds, steroids, triterpenoids and glycoside compounds, of which flavonoids appeared to be present in the highest quantity in all the four different extracts (Ramesh et al., 2013). The chemical constituents of its barks have also been reported to include flavonoids (Ata et al., 2009). These phytochemicals are known to possess antioxidant, anti-inflammatory, and anti-microbial properties which are responsible for the enhanced rate of wound healing observed (Shah & Amini-Nik, 2017).\n\n\nConclusion\n\nThe extracts from V. pinnata leaves showed the ability to scavenge free radicals, with the ethyl acetate extract being the most potent. All extracts except for the methanol extract also exhibit species-specific antibacterial activities, with the ethyl acetate extract also being the most potent. Modest wound healing capabilities are also observed in the V. pinnata treated excision wound. Study limitations include our inability to determine the relative absorption of the plant extract into the excision wound tissue site, which may have an effect on the wound closure. Further optimization of the plant extract also needs to be carried out to elucidate factors such as toxicity and effective doses for human use.\n\nWe have focused on the effects of V. pinnata extract on an excision wound, the study could be extended to include other wound models such as burns and incision wounds in order to ascertain the plant extract’s wound healing capabilities.\n\n\nData availability\n\nFigshare: Evaluation of antioxidant, antibacterial and wound healing activities of Vitex pinnata. https://doi.org/10.6084/m9.figshare.11369973.v1 (Shafie et al., 2019)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nAbdullah NA, Ja'afar F, Yasin HM, et al.: Physicochemical analyses, antioxidant, antibacterial, and toxicity of propolis particles produced by stingless bee Heterotrigona itamafound in Brunei Darussalam. Heliyon. 2019; 5(9): e02476. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndersson DI, Hughes D: Antibiotic resistance and its cost: is it possible to reverse resistance? Nat Rev Microbiol. 2010; 8(4): 260–271. PubMed Abstract \| Publisher Full Text\n\nAruoma OI: Free radicals, oxidative stress, and antioxidants in human health and disease. J Am Oil Chemist's Soc. 1998; 75(2): 199–212. Publisher Full Text\n\nAta A, Mbong N, Iverson CD, et al.: Minor chemical constituents of Vitex pinnata. Nat Prod Commun. 2009; 4(1): 1–4. PubMed Abstract \| Publisher Full Text\n\nAwang-Jamil Z, Basri AM, Ahmad N, et al.: Phytochemical analysis, antimicrobial and antioxidant activities of Aidia borneensis leaf extracts. J Appl Biol Biot. 2019; 7(5): 92–97. Publisher Full Text\n\nBarchan A, Bakkali M, Arakrak A, et al.: The effects of solvents polarity on the phenolic contents and antioxidant activity of three Mentha species extracts. Int J Curr Microbiol Appl Sci. 2014; 3(11): 399–412. Reference Source\n\nBasri AM, Taha H, Ahmad N: A review on the pharmacological activities and phytochemicals of Alpinia officinarum (Galangal) extracts derived from bioassay-guided fractionation and isolation. Pharmacogn Rev. 2017; 11(21): 43–56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBelyagoubi L, Belyagoubi-Benhammou N, Coustard JM: Effects of extraction solvents on phenolic content and antioxidant properties of Pistacia atlantica.Desf fruits from Algeria. Int Food Res J. 2016; 23(3): 948–953. Reference Source\n\nBurkill I, Birtwistle W, Foxworthy F, et al.: A dictionary of the Economic Products of the Malay Peninsula (2nd ed.). Kuala Lumpur: Ministry of Agriculture and Co-operatives, Kuala Lumpur. 1966. Reference Source\n\nDiris MN, Basri AM, Metali F, et al.: Phytochemicals and antimicrobial activities of Melastoma malabathricum and Melastoma beccarianum leaf crude extracts. Res J Phytochemistry. 2017; 11(1): 35–41. Publisher Full Text\n\nGoh MP, Basri AM, Yasin H, et al.: Ethnobotanical review and pharmacological properties of selected medicinal plants in Brunei Darussalam: Litsea elliptica, Dillenia suffruticosa, Dillenia excelsa, Aidia racemosa, Vitex pinnata and Senna alata. Asian Pac J Trop Biomed. 2017; 7(2): 173–180. Publisher Full Text\n\nMartin P, Leibovich SJ: Inflammatory cells during wound repair: the good, the bad and the ugly. Trends Cell Biol. 2005; 15(11): 599–607. PubMed Abstract \| Publisher Full Text\n\nNagar HK, Srivastava AK, Srivastava R, et al.: Pharmacological Investigation of the Wound Healing Activity of Cestrum nocturnum (L.) Ointment in Wistar Albino Rats. J Pharm (Cairo). 2016; 2016: 9249040. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNascimento GG, Locatelli J, Freitas PC, et al.: Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. Brazilian J Microbiol. 2000; 31(4): 247–256. Publisher Full Text\n\nRamesh S, Rajasekar K, Venkata Raju RR: Preliminary phytochemical studies on leaves of Vitex species (Verbenaceae), used by the local adivasi communities of Andhra Pradesh. World J Pharm Pharm Sci. 2013; 2: 6143–6150. Reference Source\n\nRashed KN: Antioxidant activity of different extracts of Vitex agnus-castus (L.) and phytochemical profile. Res Pharm. 2013; 3(6): 1–5. Reference Source\n\nSahu P, Barik B: Antipyretic and wound healing activity of aqueous extract of leaves of Vitex pinnata. Indian Drugs. 2007; 44(7): 532–534. Reference Source\n\nSaklani S, Mishra AP, Chandra H, et al.: Comparative evaluation of polyphenol contents and antioxidant activities between ethanol extracts of Vitex negundo and Vitex trifolia L. leaves by different methods. Plants (Basel). 2017; 6(4): pii: E45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShafie NA, Suhaili NA, Taha H, et al.: Evaluation of antioxidant, antibacterial and wound healing activities of Vitex pinnata. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.11369973.v1\n\nShah A, Amini-Nik S: The Role of Phytochemicals in the Inflammatory Phase of Wound Healing. Int J Mol Sci. 2017; 18(5): pii: E1068. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShi HX, Lin C, Lin BB, et al.: The anti-scar effects of basic fibroblast growth factor on the wound repair in vitro and in vivo. PLoS One. 2013; 8(4): e59966. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStankovic N, Mihajilov-Krstev T, Zlatkovic B, et al.: Antibacterial and antioxidant activity of traditional medicinal plants from the Balkan Peninsula. NJAS-Wagen J Life Sc. 2016; 78: 21–28. Publisher Full Text\n\nSuksamrarn A, Sommechai C: Ecdysteroids From Vitex pinnata. Phytochemistry. 1993; 32(2): 303–306. Publisher Full Text\n\nUmachigi SP, Jayaveera KN, Ashok Kumar CK, et al.: Studies on wound healing properties of Quercus infectoria. Trop J Pharm Res. 2008; 7(1): 913–919. Publisher Full Text\n\nVats S: Antioxidant activity of callus culture of Vigna unguiculata (L.) Walp. Researcher. 2012; 4(6): 22–24. Reference Source\n\nZhang Q: Effects of extraction solvents on phytochemicals and antioxidant activities of walnut (Juglans regia L.) green husk extracts. Eur J Food Sci Tech. 2015; 3(5): 15–21. Reference Source" }	[ { "id": "63436", "date": "20 May 2020", "name": "Yoke Keong Yong", "expertise": [ "Reviewer Expertise Natural products", "Complementary and alternative medicine", "vascular biology", "cancer biology", "non-communicable 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\nSection: Introduction\nProblem statement not clear.\n\nSection: Methods\nSuggest to add another section on “chemicals & reagents”, the authors should list all the chemicals and reagents used in this study by clearly stating the brand, company, and country.\n\nSection: Plant materials and preparation of extracts\nKindly provide a voucher specimen of the plant.\n\nSection: Results and discussion\nKindly provide yield results.\n\nCaption for all the results (Table and Figure incomplete).\n\nSection: Results and discussion – Antioxidant activities\nAuthors stated and cited phenolic content increased with the increasing polarity of solvent and extraction of total flavonoids significantly increased in polar solvents, which might contribute to the antioxidant activity by Barchan et al., 2014 and Zhang, 2015. However, in the current study ethyl acetate showed to have higher antioxidant activities compared to methanol where methanol polarity is higher than the ethyl acetate. Kindly justify this.\n\nSection: Results and discussion – Antibacterial activity\nThe authors should include the results for a positive control (reference drug), as it enables a comparison with the other treatment group.\n\nIn addition, there is no statistical analysis provided.\n\nThe concentration of the reference drug should be provided.\n\nSection: Results and discussion – Wound healing\nKindly provide details of the figures and tables, such as SEM or SD.\n\nKindly re-run the statistical analysis for the data of Figure 3 (especially Day 14) as the Control group showed big SEM or SD, however, there was a significant difference comparing Control and High dose at P<0.01.\n\nSample size of animal=3, which I doubt is enough for statistical analysis, minimum should have been at least 6 for each group.\n\nKindly justify where is the positive group.\n\nThe authors stated the study demonstrated dose dependent effect, however, I can’t see any dose dependent data. Kindly justify.\n\nLack of labeling in Figure 4.\n\nOther comments:\nTukey’s test is to compare all group, however, the way the author presented was more to Dunnet, which was compared to Control only\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?\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? Yes", "responses": [ { "c_id": "5796", "date": "07 Aug 2020", "name": "NORHAYATI AHMAD", "role": "Author Response", "response": "Section: Introduction Problem statement not clear. Response: Many thanks to the reviewer for kindly reviewing our manuscript. We have revised the introduction by adding the sentences below: Although V. pinnata is a popular ethnomedicinal plant, scientific studies are lacking to validate its pharmacological properties. It is not clear if this plant has antioxidant, antimicrobial and wound healing properties. This study is interested to determine if V. pinnata could be a good source of antioxidants. Therefore, this study also aims to screen its antibacterial activity against several bacterial strains. Section: Methods Suggest to add another section on “chemicals & reagents”, the authors should list all the chemicals and reagents used in this study by clearly stating the brand, company, and country. Response: We have added a subsection titled “Chemicals and reagents”.Section: Plant materials and preparation of extracts Kindly provide a voucher specimen of the plant. Response: We have revised the manuscript by adding: “Voucher specimen of V. pinnata (catalog ID S00059) is available in UBD Herbarium (http://ubdherbarium.fos.ubd.edu.bn/).” Section: Results and discussion Kindly provide yield results. Response: We have added a subsection titled “Extraction yields”. Caption for all the results (Table and Figure incomplete). Response: We have added the caption “Measurement of wound area” for Figure 1. Section: Results and discussion – Antioxidant activities Authors stated and cited phenolic content increased with the increasing polarity of solvent and extraction of total flavonoids significantly increased in polar solvents, which might contribute to the antioxidant activity by Barchan et al., 2014 and Zhang, 2015. However, in the current study ethyl acetate showed to have higher antioxidant activities compared to methanol where methanol polarity is higher than the ethyl acetate. Kindly justify this. Response: We have revised the manuscript by adding: “However, in this study, ethyl acetate had higher antioxidant activity compared to the more polar methanol. This could probably mean that certain non-polar compounds, which probably dissolved better in ethyl acetate, might also contribute to the higher antioxidant activity.Section: Results and discussion – Antibacterial activity The authors should include the results for a positive control (reference drug), as it enables a comparison with the other treatment group. Response: The results for the positive control is included but one has to click Table 2 and underneath the table, one will see “positive control (streptomycin sulphate) showed inhibition zones ranging from ~20 to 30 mm”. In addition, there is no statistical analysis provided. Response: We prefer not to provide statistical analysis as this is only meant as a screening test. Further method such as broth microdilution method has to be carried out to determine which extract is more potent in its antibacterial activity. The concentration of the reference drug should be provided. Response: We have added “20 mg/ml” in the methods. Section: Results and discussion – Wound healing Kindly provide details of the figures and tables, such as SEM or SD. ResponseThe ± SEM values have been included in the respective legends according to the data set. Kindly re-run the statistical analysis for the data of Figure 3 (especially Day 14) as the Control group showed big SEM or SD, however, there was a significant difference comparing Control and High dose at P<0.01. ResponseWe have rectified the error on our part in the initial figure which was during drawing of the error bars. According to Table 3, the +/- SEM was 0.3. This has been rectified in the latest version of Figure 3. Sample size of animal=3, which I doubt is enough for statistical analysis, minimum should have been at least 6 for each group. ResponseWe have not conducted power analysis to determine the minimal number of animals for the study and have used the number of animals based on previous reported studies of similar nature. We agree that having more animals per group would produce more sound statistical outcomes. We agree that this is a major shortcoming on our study. However we do feel that the results of our findings are noteworthy. Kindly justify where is the positive group. ResponseIn this study we have not included a positive control group. The high dose treated animals showed a higher percentage of wound contraction at Day 14 compared to the normal untreated and group and have shown a significant difference. The authors stated the study demonstrated dose dependent effect; however, I can’t see any dose dependent data. Kindly justify. ResponseThe dose dependent effect in the context of our work is the effects that we have observed for the high dose (50% (w/w) leaf extracts and low dose group (10% (w/w) leaf extracts where there is a difference in the percentage contractions of the wound. Lack of labeling in Figure 4. Response A labelled figure is now provided. Other comments: Tukey’s test is to compare all group, however, the way the author presented was more to Dunnet, which was compared to Control only ResponseThe study analysis was conducted to compare the wound contractions of treated vs control." } ] }, { "id": "67298", "date": "20 Jul 2020", "name": "Fitmawati Fitmawati", "expertise": [ "Reviewer Expertise Herbal Medicine", "Plant Taxonomy" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGenerally, this article provides novelty information in the field of natural antioxidant sources from the tropics. The method used is good and detailed. The results have been explained well, as well as the discussion. However, the abstract did not convey a compelling reason to underlie this study, so the impression of this study was flat. I suggest writing three sentences that show the reasons for the importance of this research in the abstract and in the introduction. Then, explain fluently the strong relationship between the elements of the title, background, objectives, methods, results, and conclusions.\n\nFor example abstract : Vitex pinnata is one of the many plants known for its ethnomedicinal properties (choose the strong sentence for the first to give a strong reason in choosing this research).\nIn the same case in the introduction, make sure the word chosen is a strong word to underlie this research and is described logically and fluently.\n\nIs the work 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": "5797", "date": "07 Aug 2020", "name": "NORHAYATI AHMAD", "role": "Author Response", "response": "Many thanks to the reviewer for kindly reviewing our manuscript. We have changed the abstract to: “Vitex pinnata is a popular ethnomedicinal plant but scientific studies to validate its pharmacological properties are lacking for this plant.” We also have revised the introduction by adding the sentences below: Although V. pinnata is a popular ethnomedicinal plant, scientific studies are lacking to validate its pharmacological properties. It is not clear if this plant has antioxidant, antimicrobial and wound healing properties. This study is interested to determine if V. pinnata could be a good source of antioxidants. Therefore, this study also aims to screen its antibacterial activity against several bacterial strains." } ] } ]	1	https://f1000research.com/articles/9-187
https://f1000research.com/articles/9-942/v1	07 Aug 20	{ "type": "Software Tool Article", "title": "Dictionary of disease ontologies (DODO): a graph database to facilitate access and interaction with disease and phenotype ontologies", "authors": [ "Liesbeth François", "Jonathan van Eyll", "Patrice Godard", "Jonathan van Eyll", "Patrice Godard" ], "abstract": "The formal, hierarchical classification of diseases and phenotypes in ontologies facilitates the connection to various biomedical databases (drugs, drug targets, genetic variant, literature information...). Connecting these resources is complicated by the use of heterogeneous disease definitions, and differences in granularity and structure. Despite ongoing efforts on integration, two challenges remain: (1) no resource provides a complete mapping across the multitude of disease ontologies and (2) there is no software available to comprehensively explore and interact with disease ontologies. In this paper, the DODO (Dictionary of Disease Ontology) database and R package are presented. DODO aims to deal with these two challenges by constructing a meta-database incorporating information of different publicly available disease ontologies. Thanks to the graph implementation, DODO allows the identification of indirect cross-references by allowing some relationships to be transitive. The R package provides several functions to build and interact with disease networks or convert identifiers between ontologies. They specifically aim to facilitate the integration of information from life science databases without the need to harmonize these upfront. The workflow for local adaptation and extension of the DODO database and a docker image with a DODO database instance are available.", "keywords": [ "disease ontologies", "diseases", "phenotypes", "database", "identifiers" ], "content": "Introduction\n\nOntologies have been developed to structure, classify, and describe diseases1–3. As ontologies were independently created to support different biomedical databases dealing with genetics, treatments, and demographics, they differ in granularity and organization and define diseases in different ways2–8. While this stimulated the construction of integrated biological knowledgebases, the use of independent, ontology-specific identifiers, heterogeneous decisions on disease definitions, and the inherent presence of errors complicates integrating disease ontologies6,8. In addition, the navigation of these large integrated knowledgebases, with often an inherently complicated data model, is difficult for most, non-expert users4,6,9.\n\nEfforts have been made to establish new integrative disease ontologies8,10,11. Using semantic similarity, the Monarch Disease Ontology (MonDO) aggregates different sources including OMIM, Orphanet, NCiT, GARD, DO, and MF10,11. The Disease Ontology (DO) resource aims to standardize disease descriptions and classifications from a clinical perspective using equivalence mappings12–14. Finally, the Experimental Factor Ontology (EFO) also establishes a unified ontology (not limited to diseases) by re-using several reference ontologies that lie within its scope. It subsequently enriches these classes with additional axioms when needed (Malone et al. 2010). Currently, it combines information from OMIM, Orphanet, ICD9/10 and SNOMEDCT, HPO, UBERON, and MonDO.\n\nDespite these ongoing efforts, two bottlenecks hamper the connection of information around disease ontologies efficiently: (1) no resource provides a complete mapping across the multitude of ontologies and (2) there is no software available to comprehensively explore and interact with disease ontologies. The Dictionary of Disease Ontologies (DODO) was developed to address these two challenges. (1) While efforts such as MonDO and EFO try to integrate different disease ontologies through semantic learning and manual curation, these resources, like the different disease ontologies themselves, are currently not providing a complete mapping across all diseases ontologies8,9. In addition, the existing efforts for integration are not flexible to extend easily to proprietary disease ontologies. DODO combines the information provided by the different ontologies and allows connecting ontologies to one another, even if they don’t have direct cross-references. This is achieved by transitive mapping and carefully considering some indirect cross-reference relationships. (2) The second challenge addressed by DODO is the lack of an efficient and straightforward manner to access disease information through well-established bioinformatics platforms (such as R or python)8,15. Such access would facilitate a more flexible connection to the different life science resources and create a more complete biomedical knowledge landscape. Currently, the programmatic access provided by many ontologies often requires expertise in creating SPARQL queries and a high level of understanding of the underlying databases or data model to be able to generate more complex queries4,8,9. The DODO R package allows easier access, exploration, and definition of disease concepts of interest. It can work as an intermediate layer to facilitate access and ensure exhaustive extraction of information from life science databases without the need to harmonize upfront. Here, the DODO graph database and R package are introduced, and we exemplify their added value by going through some use cases.\n\n\nMethods\n\nR version: R version 3.6.0 (2019-04-26)\n\nBioconductor version: 3.10\n\nPackage: 1.0.0\n\nIn this section, an overview of the DODO database and the R package is presented.\n\nThe data model underlying DODO aims to capture the relationship between disease and phenotypes as described across different databases (Figure 1). Disease and Phenotype are two different kinds of Concepts sharing the same properties and they are related through a has_pheno relationship. Concepts are identified by name, the concatenation of the short identifier (shortID) and database. Additional properties of a Concept are (when available) a unique canonical label, disease definition, several Synonyms, and node type. Each Concept is_in one or multiple Databases with their URL encoded in the idURL property. Therefore, the Database name can differ from the Concept database as some ontologies re-use Concept names like EFO7. If the concepts are hierarchically organized, the level captures the highest level a disease has in the ontological tree (the most general term being level 0). This information is encoded as a property of a Concept where it captures the level of the term in the original ontology. In addition, as Concepts can be re-used, the ontology-specific level is also encoded as a property of the is_in relationship. Hierarchical information is also encoded by identifying a parent Concept through an is_a relationship. The property origin is assigned to this edge and corresponds to the ontology from which the relationship is derived (this can be useful when Concept names are re-used). Former and alternative identifiers of a Concept are documented using is_alt relationship.\n\nIt is shown as an Entity/Relationship (ER) diagram: entities (concept, disease, phenotype, database) correspond to graph nodes and relationships to graph edges. ID and idx refer to a unique or indexed entity respectively. Some properties are duplicated in upper case (_up) to improve the performance of case-insensitive searches. The system node is a technical node used to document the DODO instance.\"\n\nCross-references between Concepts are managed through two types of relationships depending of the confidence put on them (more detailed explanation below). The is_xref relationship is considered for more trusted relationships compared to is_related relationship. Each cross-reference (is_xref and is_related) between two Concept nodes is recorded as two edges, each in one direction. A Concept can have multiple cross-reference relationships to nodes of the same database. This ambiguity (one-to-many) is captured by the property FA (forward ambiguity) on a cross-reference edge and captures the number of cross-references to the same Concept database. The BA (backward ambiguity) property of the edge is defined symmetrically as the FA property of the edge going in the opposite direction. Both types of cross-reference edges and forward and backward ambiguities are used to define the relations used for transitivity mapping as explained below.\n\nA DODO instance is built on data from external resources that should be pre-processed to organize the information. This work was done for several public ontologies and the scripts can be found in the corresponding GitHub repositories (Table 1).\n\nAll software under GPL-3.\n\nA R markdown document showing how to construct a new instance is provided alongside with a set of scripts to load and feed a Neo4j instance. These are not exported to avoid confusing the user when querying the database. The different steps to construct a new DODO Neo4j instance are briefly described below:\n\n1. Structuring and harmonize the information derived from each ontology\n\n2. Combine the information obtained from the different ontologies and identify any duplicate or missing data\n\n3. Start a new Neo4j instance and load data model\n\n4. Information is imported into Neo4j by type. First, information around database nodes and concept nodes is imported. Next, information on the different relationships are loaded into the instance (cross-references, parent/child, alternative identifiers, phenotype mappings). For cross-reference identifiers, the type of edge is defined based on (see Extended Data16) and subsequently imported into Neo4j. After import, the forward and backward ambiguity is calculated on each edge and assigned as property values to that edge.\n\nThe DODO instance build using the workflow described above is provided as a Docker image17: https://hub.docker.com/repository/docker/elysheba/public-dodo (tag: 02.04.2020). This instance is built on information from the following disease ontologies listed in (Table 1).\n\nDODO contains information on 54 different disease ontologies (Figure 2). There are 418,881 disease nodes and 18,354 phenotype nodes present in the database. 92,300 disease nodes have no recorded is_xref or is_related relationships across ontologies. The number of edges per relationship type is listed in Table 2.\n\n30 ontologies have less than 100 entries in DODO are summarized as ’other’.\n\nThe database is implemented in Neo4j which uses the Cypher query language18. A DODO R package was developed to interact with and provide higher level functions to query the Neo4j graph database based on the data model described above19.\n\nThe minimal system requirements are:\n\nR ≥ 3.6\n\nOperating system: Linux, macOS, Windows\n\nMemory ≥ 4GB RAM\n\nThe graph database has been implemented with Neo4j 3.5.1418 with the apoc.path.expand procedure 3.5.0.11. The DODO R package uses the following packages:\n\ndplyr20\n\ntibble21\n\nneo2R22\n\nrlist23\n\nstringr24\n\nreadr25\n\nvisNetwork26\n\nshinythemes27\n\nDT28\n\nigraph29\n\nshiny30\n\nThe DODO R package combines several functions to construct, interact, and explore the relationships between disease and phenotype identifiers. These can be divided in five different scopes (see Extended Data16):\n\nDisease network functions: these functions allow the user to build a disease network based on their relationships in the graph database: cross-reference, hierarchical information, and phenotypes. These functions include ways to extend, filter, split or combine disease networks. The information on diseases and phenotypes and their relationships is structured as a S3 disease network (disNet) object. It captures all information (disease node information, hierarchical information, phenotype information, alternative identifiers, and cross-reference information) around a disease.\n\nVisualization and exploration: these functions allow the exploration and visualization of relationships between disease and phenotype identifiers by querying them or providing a disease network.\n\nConversion: this category of functions converts a list of disease and phenotype identifiers across ontologies or concepts based on the data model and provided parameters. Conversion can also include indirect relationships across ontologies using transitive mappings.\n\nConnection and low level interactions: these functions are technical helpers for establishing and managing connection with a DODO graph database. These functions also return information on the content of the current database and allow to directly send cypher queries to the database.\n\nData information: these functions give access to content information such as the list of original databases, concept description and reference URL and allow ontology dump.\n\nAmbiguity of cross-reference relationships Disease identifiers within the different ontologies are annotated with cross-reference relationships to connect to independent biomedical databases. However, no ontology provides a complete mapping across all existing ontologies8,9. Efforts to create an integrated ontology such as MonDO and EFO are continuously expanding to enrich their mappings but are currently not exhaustive and will lack mappings to proprietary ontologies. Here we propose the use of transitive mappings to extend the cross-reference information and connect ontologies (and biomedical resources) that lack direct relationship(s). This is exemplified in Figure 3, where transitivity mappings is needed to connect the initial MONDO identifier to a MeSH identifier using the indirect cross-reference relationship to the EFO and ORPHA node.\n\nMost mappings are unambiguous (one concept in an ontology is related to only one concept in another ontology); however, some concepts map to many similar concepts within the same ontology. This is conceptually visualized in Figure 3 where the MONDO identifier maps to 2 ORPHA identifiers.\n\nA real example is provided in Figure 4 with focus on the “Coffin-Lowry syndrome” (ORPHA: 192), a rare syndrome affecting brain and skeleton development. It relates to many disease identifiers, that often share similar disease definitions in an unambiguous or one-on-one manner. These relationships are valuable for transitive mapping as they extend the initial node to very similar nodes in other ontologies. However, the cross-reference relationship to ICD10 deals with a very broad term of “Congenital malformation syndromes predominantly affecting facial appearance” (ICD:Q87.0). This identifier is highly ambiguous as it has 285 additional direct cross-reference relationships to other disease identifiers. The use of this type of indirect relationships needs to be carefully considered. This ambiguity is a consequence of the inter-ontology differences in concept definitions and precision where some cross-reference edges connect identifiers that are not equivalent. Ontologies such as MonDO or EFO use a greater granularity in disease definitions than others like ICD10 or ICD9. If cross-reference edges are all considered equal without taking this distinction into account, it would be detrimental to the relevance of the conversion as it will return numerous more distantly related concepts when traversing these edges. Therefore, such relationships need to be avoided for transitive mappings.\n\nThe network shows equivalent cross-reference relations and ambiguous cross-reference relation to ICD10:Q87.0. This ICD10 disease identifier connects to 283 additional disease identifiers. The colors refer to different databases. Is_xref and is_related edges are indicated in blue and orange respectively. The arrows on the edge refer to the backward ambiguity: the identifier at the arrow destination has only one cross-reference in the database of the identifier at the arrow start. A double arrow indicates an unambiguous mapping between the 2 databases for the 2 identifiers in both directions. An edge should be considered transitive only in the direction of the arrow otherwise it can only be the final edge of a conversion path.\n\nTo prevent these inaccurate conversions, we propose the use of backward ambiguity filtering. First, the forward ambiguity of a cross-reference edge needs to be quantified and it is the number of relationships where a node maps to several similar concepts within the same ontology. The value of the forward ambiguity is shown for the example in Figure 4 on each cross-reference edge between the nodes. A forward ambiguity greater than one indicates that the original concept is likely more general than the concepts it maps to. By following the relationship in the other direction, this ambiguity value is considered as the backward ambiguity. Similarly, a backward ambiguity greater than one, indicates that the original concept is probably more precise than the concepts it is mapped to (Figure 4). Applied to our example, the filtering on ambiguity will prevent traversing through the ambiguous ICD10 when using the transitivity mechanisms and return only cross-reference identifiers close to the original node.\n\nDefining subtypes of cross-reference edges The filter on backward ambiguity improves greatly the accuracy of conversions. However, some inaccurate mappings may still be present due to higher general ambiguity between specific ontologies. The maximum total ambiguity of all relationships between two ontologies (the maximum value of the sum of forward and backward ambiguities of all cross-reference edges) quantifies the symmetry of their cross-reference relationships. The heatmap in Figure 5 shows this value in a log10 scale. While many ontologies are using concepts of similar level as identified by low maximum total ambiguity, a few can be identified that are more ambiguous in their mappings. Therefore, the general trust assigned to cross-reference relationships between ontologies is captured by defining two types of cross-reference edges: (1) the is_xref edge is used for equal cross-reference relationships where the concepts relate more directly to each other (similar concept definitions); (2) the is_related edge is used for all other cross-reference edges.\n\nTo assign relations to either type, a threshold is applied on the maximum total ambiguity between two ontologies. Different thresholds between 2 and 20 have been explored taking 14 different ontologies into account. For each step the cross-reference relationships between ontologies with the maximum total ambiguity falling below the threshold are defined as is_xref and otherwise as is_related. Next, all the identifiers from an ontology are converted to identifiers from any other ontology by applying transitive mappings only on is_xref relationships with a backward ambiguity of 1 (step = NULL, intransitive_ambiguity = 1). Since the number of is_xref relationships increases with the threshold applied, the number of conversions achieved from each identifier increases as well (see Figure 6 for an example focused on the MONDO ontology).\n\nThis number depends on the cut-offs in the (total) ambiguity to define is_xref and is_related cross-reference edges on an ontology scale shown in x axis.\n\nWhen choosing a conservative and low threshold, the conversions may be more precise but will hamper the ability of the transitive mappings to identify the most relevant mappings. It may therefore not return all cross-reference identifiers whereas a too high threshold impacts strongly the number of converted nodes at the cost of some inaccurate conversions. Especially the effect of on a limited set of identifiers is strong with the return of many, more distantly related and less precise cross-reference identifiers as can be seen by looking at the tail of the boxplot shown on Figure 6.\n\nAfter having compared the results of the different ontologies, the threshold on the maximum total ambiguity value has been arbitrarily set to 4: cross-references between ontology with a maximum total ambiguity below 4 were considered as is_xref and others as is_related edges (see Extended Data16). However, two exceptions are ICD9/ICD10 and OMIM/Orphanet. Both ICD9 and ICD10 use higher level disease definitions, and therefore, these will never be connected through an is_xref edge except between themselves. OMIM and Orphanet on the other hand, use very narrow subtypes of diseases or mention specific variants. This results in a higher ambiguity as a general disease may have many relations to subtypes but as these ontologies use a higher granularity their relationships are still encoded as an is_xref edge.\n\n\nUse cases\n\nOne of the basic functionalities of DODO is the ability to convert disease and phenotype identifiers between ontologies. The conversion of identifiers is generally performed using a two-step process based on the type of cross-reference edge to traverse and ambiguity values to filter on. The first step uses transitivity on is_xref edges only to identify the high confidence identifiers mapped to the initial identifier of interest. This makes the core of cross-referenced identifiers. It is strongly recommended to limit the backward ambiguity in this transitive mapping to one (transitive_ambiguity = 1 (default)) to avoid unwilled conversion to less accurate concepts. Once the core of identifiers is established, the second step expands it by a single step using both types of cross-reference edges (is_xref and is_related) with specification on the backward ambiguity filtering (“intransitive_ambiguity” parameter).\n\nSix separate use cases can be identified for converting disease or phenotype identifiers to other ontologies or concepts:\n\nDirect conversion: only return the direct cross-references without any transitive mapping\n\nStrict indirect conversion: uses transitive mapping and applies a filter on the backward ambiguity of the last step to only return equivalent concepts. Can be used to convert between ontologies that use similar granularity to define concepts.\n\nExtended indirect conversion (default): uses transitive mapping without any filter on the backward ambiguity of the last step to return both equivalent and more broader terms. This way of conversion returns ambiguous relations, but these relations are not used for transitive mapping steps. In general, when the aim is to reach a broader concept related to the original identifiers but not move through it, it is recommended to put no filter on the backward ambiguity filtering of the final step.\n\nLoosened indirect conversion: a specific conversion procedure is created for ontologies that are less connected through is_xref edges such as ontologies like WHO’s ICD10 or ICD9. These ontologies have highly ambiguous mappings and use very broad disease definitions. The absence of is_xref cross-reference relationships restrict the utility of the transitivity mechanism when applying the standard conversion. Therefore, we recommend an additional step to the standard conversion implemented in the get_related function.\n\nConversion between concepts: convert between concepts types, i.e. from disease identifier to phenotype identifiers or vice versa.\n\nReturn deprecated identifiers: return previous version (deprecated) identifiers\n\nThe use cases will be illustrated using the Mondo identifier for epilepsy (MONDO:0005027). The conversion between concept types (phenotype to disease or vice versa) is exemplified here for one identifier; however, the conversion procedure can take multiple identifiers at once as input. Finally, a comparison of the different conversion possibilities is performed using the entire MonDO ontology.\n\nA first basic use case is conversion of the identifier of interest to direct cross-references (without any transitive mapping - parameter “step = 1”). As an example, MonDO identifier for epilepsy (MONDO:0005027) is mapped to its corresponding EFO identifier using convert_concept function.\n\nIn this first use case, only the direct relations are used to return the corresponding EFO identifiers (highlighted in red on Figure 7). Using transitive mappings is also not required for this example, as there would be no additional EFO identifiers returned by moving through indirect relationships.\n\nThe seed node, MONDO:0005027, is depicted as a yellow triangle. Subsequently, the results of the different use cases are highlighted. The results of use case 1 (direct conversion) is indicated in red. The results of use case 2 (strict indirect conversion) and 3 (extended indirect conversion) to obtain corresponding Orphanet identifiers using transitivity are indicated in blue and green, respectively. Is_xref and is_related edges are indicated in blue and orange respectively. The arrows on the edge indicate the direction mapping can occur taking into account a backward ambiguity of one. Absence of arrow on an edge indicate these relations can only be exploited when no filtering is applied (generally final step of conversion).\n\n\n\n\n\nA second use case deals with the conversion using equivalent indirect relations. The same seed MonDO identifier for epilepsy will be mapped to return the corresponding Orphanet ontology identifier. By specifying “step = NULL”, transitive mappings will be used to convert the identifier(s). To return only equivalent concepts, thereby avoiding the mapping to less precise disease concepts, backward ambiguity filtering is applied on the final step of conversion (“intransitive_ambiguity = 1”). This can be a good practice when converting between ontologies that define concept with similar granularity. As there is no direct mapping provided by the resources between MonDO and Orphanet, transitive mapping provided by DODO needs to be applied. This conversion of the seed identifier MONDO:0005027 returns one corresponding Orphanet identifier (ORPHA:166463) as shown in Figure 7 (blue node).\n\n\n\n\n\nIn some instances, you may want to define a list of disease identifiers more largely related to a disease of interest. The third use case addresses this by extending the transitivity mapping with no filtering on the final step of conversion (“intransitive_ambiguity = NULL” and “step = NULL”). The conversion of the seed identifier MONDO:0005027 to Orphanet with this setup, returns an additional Orphanet identifier (ORPHA:101993) indicated in green on Figure 7. The initial transitive mapping steps apply filtering with ambiguity equal to one via the is_xref edges (blue). The final mapping step is intransitive and will therefore return all nodes related through either an is_related or is_xref edge with no filtering on backward ambiguity. It does return ambiguous relations at the last step, but these relations are not used for transitive mapping steps. This conversion can be used to get all identifiers around a disease concept both equivalent and more broader terms or when converting from a narrower concept to a broader concept. In general, when the aim is to reach a broader concept related to the original identifiers but not move through it, it is recommended to put no filter on the “intransitive_ambiguity”.\n\n\n\n\n\nThe fourth use case deals with the specific conversion procedure that is recommended for the ontologies which are less connected through is_xref edges to the “core” ontologies in DODO (e.g. MonDO, EFO, MedGen, etc.) such as ontologies like WHO’s ICD10 or ICD9. These ontologies have highly ambiguous mappings and use very broad disease definitions. When starting the mapping from such ambiguous identifiers, the convert_concept function will not return cross-reference relationships to most other databases. It is restricted by the rules defined by transitivity mechanism. Therefore, we recommend an additional step to the standard conversion implemented in the get_related function. It performs an additional expansion step through is_related and is_xref edges before the standard conversion procedure. The ambiguity on this additional step is the same for the final step in the standard conversion procedure (use case 2 – strict indirect conversion - modified by the intransitive_ambiguity parameter equal to NULL). To illustrate the difference, we will show the conversion of the general ICD10 identifier for Epilepsy (ICD10:G40.9) to corresponding identifiers in DO. When using the standard convert_concept function, it is not possible to convert the ICD10 identifier to any DO identifier. As ICD10 is only related to other nodes via is_related edges, transitive relationships cannot be used to identify cross-references using the standard conversion. Using the get_related function, recommended for ontologies such as ICD10, does convert ICD10:G40.9 to corresponding DO identifiers through transitive relations and returns DOID:1826 (Epilepsy).\n\n\n\n\n\n\n\n\n\nThe underlying reason for this behavior and difference between the two functions is depicted in Figure 8. As there are no relationships from the ICD10 seed node that meet the criteria defined for transitive mapping (intranstivity_ambiguity = 1 and cross-reference relationships of is_xref type), the seed node cannot be mapped to the DO node of interest using the convert_concept function. The get_related function relaxes these transitive mapping criteria in the first step and allow to reach the directly related nodes connected by either type of relationships and with no constraint on backward ambiguity (green round circles in Figure 8). After this initial step, transitivity rules are applied to map the direct cross-reference identifiers to the identifier in DO (orange node in Figure 8).\n\nIs_xref and is_related edges are indicated in blue and orange respectively. The arrows on the edge indicate the direction mapping can occur taking into account the BA = 1 rule. Absence of arrow on an edge indicate these relations can only be exploited when no filtering is applied. Using the get_related functionality adds an additional step of conversion before applying the standard conversion procedure. The nodes indicated in green are those that would be returned using the standard convert_concept function. The grey nodes are those reachable by get_related function that relaxes the initial step of transitivity mappings through is_related edges. The retrieval of corresponding DO identifiers for ICD10:G40.9 returns DOID:1826 (indicated in orange).\n\nCurrently, the default conversion procedure refers to the third use case (extended indirect conversion) when transitivity mappings are used to extend through cross-reference relationships and no filtering is applied to the final step of extension through the network (default parameters: step = NULL, intransitive_ambiguity = 1). This conversion can be used to get all identifiers around a disease concept and it is the approach to use when converting from a narrower concept to a broader concept. Additional filtering can then subsequently be put in place to allow adaptation for specific use cases. However, for the first step using transitivity mapping on is_xref edges it is strongly recommended to use the default filtering on ambiguity by limiting (backward) ambiguity to one.\n\nConversion can also be used to convert between concepts types, i.e. from disease identifier to phenotype identifiers or vice versa. This conversion is achieved by the same convert_concept function that will leverage has_pheno relationships. Practically, it is handled in two phases. The first phase uses the transitivity mechanism as detailed above to connect disease identifiers to phenotype identifiers even when no direct has_pheno relation is available. This initial step takes the same options as listed above to convert identifiers within the same concept (this step can be avoided by using parameter “step = NA”). The second phase converts identifiers between concepts by returning phenotype or disease nodes related to the original identifiers (including the converted identifiers obtained in the first phase) with the possibility to return direct and/or indirect relations with the parameter “step”.\n\n\n\n\n\n\n\n\n\nFinally, conversion can also be used to return previous version (deprecated) identifiers when these are available.\n\n\n\n\n\nThe different use cases were applied to only one identifier to show in more detail the conversion procedure. However, the conversion procedure is designed to take multiple identifiers as input. Here, the first three different approaches outlined in the previous section are applied to convert all 21,653 identifiers in the MonDO ontology to their corresponding identifiers in EFO, MesH, and DO. The mapping outcomes are summarized in Table 3.\n\n1Number of unique MonDO identifiers with a conversion\n\n2Number of unique converted identifiers in the targeted ontology\n\n3Distribution of the number of conversions returned per MonDO identifier.\n\n4Number of MonDO identifiers with ambiguous conversions\n\n5Conversion as performed by use case 1: direct conversion (see above), the parameter is step = 1\n\n6Conversion as performed by use case 2: strict indirect conversion (see above), the parameters are step = NULL and intransitive_ambiguity = 1\n\n7Conversion as performed by use case 3: extended indirect conversion (see above), the parameters are step = NULL and intransitive_ambiguity = NULL)\n\n\n\n\n\nExcept for Disease Ontology (DO) (which is included by MonDO while constructing their ontology based on semantic similarity), the majority of MonDO identifiers cannot be converted to EFO or MeSH identifiers. However, compared to direct mapping of encoded relationships (use case 1 – direct conversion), the use of transitive mapping allows the conversion of 20% additional MonDO identifiers (use case 2 – strict indirect conversion). Enabling the extension to broader disease concepts (use case 3 – extended indirect conversion), still increases, by design and as expected, the number of mappings between two ontologies.\n\nIn parallel, the average ambiguity in mappings also increases with the different use cases (Table 3 - column 3). While it varies strongly across the different ontologies, the ambiguity is mostly minor for most identifiers (based on median and mean). Still, a limited set of mappings is strongly affected by ambiguity with as many as 294 corresponding DO identifiers and 91 corresponding MeSH identifiers for “autosomal dominant non-syndromic deafness” (MONDO:0019587). This identifier could not be mapped to EFO. Looking into this identifier in more detail shows that it only has six direct cross-references among which one DO identifier (DOID:0050564) (Figure 9). However, these direct cross-reference identifiers themselves report a multitude of cross-reference identifiers encoding various subtypes of the condition. As mentioned before, the mappings are derived directly from the original resources. The observed ambiguity after transitive mapping highlights disease areas that are heterogeneously defined across ontologies. This ambiguity is naturally more prevalent for those indications with a lot of reported subtypes reported in different ontology and/or the ontology using a higher level of granularity used to define diseases.\n\nBuilding a disease network. While conversion facilitates connecting biomedical resources directly, another possibility provided by DODO is the exploration of diseases and their relationships as a disease network. Contrary to conversion, a network retains all relationships as they are encoded in the DODO graph database. In this use case, we will show how to construct such a disease network and which functionalities are available to interact with a network.\n\n\n\n\n\nExtension through different relationships. After the construction of a disease network, it is likely that it doesn’t contain the complete information on that particular disease of interest. The extend_disNet function enriches the disNet and extends it to cross-reference identifiers, child/parent terms, annotated phenotypes/disease, and/or alternative identifiers when available. In concordance with the conversion procedure, extension follows the same two-step approach using transitive mapping on is_xref edges followed by a final one-step extension on any cross-reference relationship taking filtering on backward ambiguity into account. To perform this extension the same parameters can be supplied with similar aims as for the conversion procedure (see above).\n\n\n\n\n\nThe disease network gathers 488 disease concepts across 25 ontologies; only 251 where identified directly matching the search term (Figure 10). The additional terms were obtained through extension of both cross-references and parent/child relationships. Of specific note is the extension to (or from) phenotype information. Within one extension all different parameters (xref, child, parent, alt, and disease/phenotype) can be supplied with the exception that it is not possible to extend to both disease and phenotype simultaneously. In contrast with the conversion procedure, it does not use the transitivity mechanisms but rather takes all the diseases within the network and returns any associated phenotypes that can be obtained through the has_pheno relationship.\n\nThis disNet is subsequently extended to return all cross-reference identifiers and child terms using the extend_disNet function (orange nodes) (parameters relations = c(‘xref’, ‘child’) and intransitive_ambiguity = 1 to return only equivalent identifiers)\n\n\n\n\n\nExplore a network of diseases. DODO is built as a meta-database incorporating several disease ontologies and their listed relationships. As disease concepts and definitions are not a natural process but rather an artificial, human-biased effort, concepts might not always be clearly defined or related to each other in a straightforward manner. The different ontologies employ heterogeneous definitions, cross-reference axes are not always exact, and errors present in the original ontologies will impact DODO as well. The explore_disNet on a single disNet object returns a data table presenting information on the different identifiers present in the network. The plot function displays as a network how diseases are related to each other across the different ontologies (Figure 11).\n\nThe colors refer to different databases. Is_xref and is_related edges are indicated in blue and orange respectively. The arrows on the edge refer to the backward ambiguity and show how an edge can be traversed. When no arrow is present, it can only be reached through the final step of conversion when no filtering is present.\n\nIt may be required to review the returned network of diseases after building and/or extending it to assess whether all nodes are relevant or of interest. This process can be simplified by considering clusters of cross-references (nodes dealing with similar concepts) using the cluster_disNet functionality.\n\n\n\n\n\n\n\nInstead of reviewing each node, the different cross-reference clusters can be reviewed to identify those of interest while using the relationships between nodes to handle equivalent nodes simultaneously without the need to review them separately. A summary of the clusters can be visualized using the explore_disNet function and the output is shown in Table 4. For a list of disease networks created after clustering the explore_disNet functionality summarizes information on the different clusters, provides information on the size of each cluster and presents a tag identifier information. This tag identifier is identified as the node with the highest level in the ontology and a label available. If multiple identifiers have the same level, the tag one is picked on alphabetical order of the label. Summarizing disease networks using cluster of cross-reference edges also allows the revision of identifiers that have no label information attached.\n\nConnecting to external resources. The aim of DODO is to facilitate the connection with external resources. As described above, DODO allows the creation of a structured disease network around diseases of interest and their relationships and information. The use of this disease network facilitates the connection and exploration of different biomedical knowledge resources as it also allows tracing their connections more easily.\n\nIt is exemplified below by connecting two different external resources: ClinVar (release 2020-03) and CHEMBL (release 25) using “amyotrophic lateral sclerosis” (ALS) as an example31,32. We start by building a disease network around the term “amyotrophic lateral sclerosis” and extending through both cross-reference and parent/child relationships as described in the previous section. For comparison, CHEMBL and ClinVar are queried directly using the same search term. Each of these resources uses a different ontology as reference. CHEMBL uses both EFO and MeSH to annotate compounds to indication. ClinVar uses a variety of ontologies to connect variants and diseases, such as SNOMEDCT, MedGen, Orphanet or OMIM.\n\nThrough the use a disease network, 96 unique compounds were identified in CHEMBL for ALS connected to four different disease identifiers (Table 5). The same set of compounds is identified by querying the resource directly, demonstrating the performance of DODO to properly map those different ontologies. One associated disease is missing in CHEMBL, namely the identifier “ORPHA:98756”. This term was identified as a child term of ‘EFO:0001356’ with the extension of the disNet using DODO and providing more granularity (Figure 12). The disease can not be identified using a free-text query in CHEMBL directly as it’s labelled ‘Spinocerebellar ataxia type 2’. However, while different resources such as Monarch Initiative and EFO report ALS as a parent term, it is unclear whether this can be considered as ALS disease. OMIM does report a genetic overlap between spinocerebellar ataxia and amyotrophic lateral sclerosis type 13. The information integrated in the DODO graph database is based on the original information provided by ontologies such as EFO and MonDO without any additional curation. Errors in disease definitions and provided mappings will inherently be present in these ontologies and will therefore persist within DODO as well. While it was not within the initial scope of DODO, it may help to assess and identify underlying issues present in the ontologies.\n\nThe term ‘ORPHA:98756’ was identified as a child term of ‘EFO:0001356’ through extension using is_a edge (green edges). Cross-reference edges are indicated in blue. The arrows on edges refer to the direction an edge can be traversed.\n\nFor the ClinVar resource associating gene variants to diseases, all 105 unique Entrez gene variants returned querying ClinVar directly for “amyotrophic lateral sclerosis” are also identified using a network of diseases as a query start. However, an additional variant was reported for “Inclusion body myopathy with early-onset Paget disease with or without frontotemporal dementia 2” (ClinVar:18286). This identifier was found by extending through cross-references edges using transitivity mapping starting from “ORPHA:52430” (“Inclusion body myopathy with Paget disease of bone and frontotemporal dementia” and “Pagetoid amyotrophic lateral sclerosis” as a synonym (Figure 13)). This disease identifier is not an actual ALS disease but rather another neurodegenerative disorder related to frontotemporal dementia. This example highlights the necessity of reviewing the queried disease not only when using a network of diseases, but the same need remains when querying resources directly. Using DODO, it is possible to use underlying disease relationships to cluster diseases and identify groups of identifiers with similar or related concepts (Table 4). Particular clusters that are outside of the scope can be dropped and a more precise network of diseases returned to connect to external knowledgebases. The reviewed network of diseases no longer includes identifiers outside of the scope and identifies the same set of 105 unique Entrez gene identifiers compared to querying the ClinVar resource directly. For CHEMBL, the results remain the same. The ability to apply use and review easily disease networks should facilitate the integration of biomedical resources.\n\nThe color of the nodes refers to the ontology, the is_xref edges are indicated in blue and is_related edges indicated in orange. The arrows on the edge refer to the backward ambiguity and show how an edge can be traversed. When no arrow is present, it can only be reached through the final step of conversion when no filtering is present.\n\n\n\nTracing connections. Understanding the relation between disease identifiers obtained when querying a resource directly through a search term is not a trivial thing. The question remains whether these identifiers are dealing with the same disease concepts. An additional feature from connecting resources using a network of diseases, is the possibility to identify if and how diseases returned from each resource are connected to each other. This does not only allow a better understanding of disease, but also facilitates downstream analyses. Figure 14 shows the original ALS extended and reviewed network with disease identifiers matching in CHEMBL (orange) and those matching in ClinVar (blue). As both resources use different ontologies as references, there is a necessity to use cross-reference to understand their relationship to each other. Indirect relationships are used and recorded when extending and can facilitate the understanding and integration of different biological resources.\n\nThe colour of the nodes refers to the disease identifiers that are also identified through a direct query in CHEMBL (orange) and ClinVar (blue). The edges between the nodes capture the is_xref and is_related relationship in blue and orange respectively.\n\n\nConclusions\n\nDisease ontologies have allowed a more formal classification of diseases. They facilitate the integration of biological databases thereby increasing disease information usage and supporting the development of novel treatments. However, efforts to integrate biological databases or the ontologies directly are complicated by ontology-specific identifiers, heterogeneous decisions on disease definitions, and the inherent presence of errors. Despite ongoing integration efforts, we identified two remaining challenges that prevent seamless integration of different databases based on disease ontologies:\n\nCurrently no resource provides a flexible and complete mapping across the multitude of disease ontologies\n\nThere is no software available to comprehensively explore and interact with disease ontologies\n\nDODO aims to tackle these two challenges by constructing a meta-database containing information on disease identifiers and their relationships across different ontologies. Through well-defined and controlled transitivity mechanisms, the combined information across resources can be used dynamically to identify indirect cross-references. The R package contains several functions to build and interact with disease networks or convert concept identifiers between ontologies. The workflow to construct a custom, local DODO database is provided with the intent to allow adaptation. A docker image with the presented ontologies is provided for convenience.\n\nDODO helps clarifying and defining conditions of interest in addition to help in the understanding of relationships between disease concepts. It improves accessibility of disease ontologies for a standard user. In addition, connecting different biomedical knowledge resources through a disease network facilitates the integration of all this information. It also ensures these resources are queried transparently using equivalent identifiers of the disease of interest. In addition, it also allows visualizing the connection between these resources directly.\n\nThrough the aggregation of different ontologies and their mappings, DODO facilitates the generation of exhaustive descriptions of disease landscapes. The code to build and query DODO is provided under open source license to allow further improvement by other developers.\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\nZenodo: Extended data for publication \"Dictionary of disease ontologies (DODO): a graph database to facilitate access and interaction with disease and phenotype ontologies\", https://doi.org/10.5281/zenodo.392221016\n\nThis project contains the following extended data:\n\nTable 1: List of all functions available in DODO R package with description and scope details.\n\nTable 2: List of ontologies among which the cross-reference relations are encoded as is_xref\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\nSource code for DODO is available at: https://github.com/Elysheba/DODO\n\nArchived source code as at time of publication: https://doi.org/10.5281/zenodo.392214333\n\nLicense: GPL-3 license\n\nDocker image of the DODO Neo4j instance is available at: https://hub.docker.com/repository/docker/elysheba/dodo (tag:02.04.2020)\n\nArchived database docker image as at time of publication: http://doi.org/10.5281/zenodo.392187434\n\nSource code (and archived source code) for parsing disease ontologies are available in Table 1.", "appendix": "References\n\nGruber TR: A Translation Approach to Portable Ontology Specifications. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMungall CJ, McMurry JA, Kohler S, et al.: The Monarch Initiative: An integrative data and analytic platform connecting phenotypes to genotypes across species. Nucleic Acids Res. 2017; 45(D1): D712–D722. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShefchek KA, Harris NL, Gargano M, et al.: The Monarch Initiative in 2019: an integrative data and analytic platform connecting phenotypes to genotypes across species. Nucleic Acids Res. 2020; 48(D1): D704–D715. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCheng L, Wang G, Li J, et al.: SIDD: A Semantically Integrated Database towards a Global View of Human Disease. PLoS One. 2013; 8(10): e75504. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchriml LM, Mitraka E: The Disease Ontology: fostering interoperability between biological and clinical human disease-related data. Mamm Genome. 2015; 26(9–10): 584–589. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYu G, Wang LG, Yan GR, et al.: DOSE: An R/Bioconductor package for disease ontology semantic and enrichment analysis. Bioinformatics. 2015; 31(4): 608–609. PubMed Abstract \| Publisher Full Text\n\nSaqi M, Lysenko A, Guo YK, et al.: Navigating the disease landscape: Knowledge representations for contextualizing molecular signatures. Brief Bioinform. 2019; 20(2): 609–623. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFrançois L: Extended data for publication \"Dictionary of disease ontologies (DODO): a graph database to facilitate access and interaction with disease and phenotype ontologies\" [Data set]. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3922210\n\nDocker Inc: Docker Community Edition. 2019. Reference Source\n\nNeo4J Inc: Neo4j Community Edition. 2020. Reference Source\n\nR Core Team: A Language and Environment for Statistical Computing. 2019. Reference Source\n\nWickham H, François R, Henry L, et al.: dplyr: a grammar of data manipulation. 2019. Reference Source\n\nMüller K, Wickham H: tibble: simple data frames. 2019. Reference Source\n\nGodard P, van Eyll J: BED:A Biological Entity Dictionary based on a graph data model [version 3; peer review: 2 approved]. F1000Res. 2018; 7: 195. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRen KK: rlist: a toolbox from non-tabular data manipulation. 2016. Reference Source\n\nWickham H: stringr: simple, consistent wrappers for common string operations. 2019. Reference Source\n\nWickham H, Hester J, François R: readr: Read Rectangular Text Data. 2018. Reference Source\n\nAlmende BV, Thieurmel B, Robert T: visNetwork: network visualization using vis.js library. 2019. Reference Source\n\nChang W: shinythemes: themes for shiny. 2018. Reference Source\n\nXie Y, Cheng J, Tan X: DT: a wrpper for the JavaScript Library \"DataTables\". 2019. Reference Source\n\nCsardi G, Nepusz T: The igraph software package for complex network research. InterJournal. Compex Systems, 1695. 2006. Reference Source\n\nChang W, Cheng J, Allaire JJ, et al.: shiny: Web Application Framework for R. 2019. Reference Source\n\nLandrum MJ, Lee JM, Benson M, et al.: ClinVar: Improving access to variant interpretations and supporting evidence. Nucleic Acids Res. 2018; 46(D1): D1062–D1067. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMendez D, Gaulton A, Bento AP, et al.: ChEMBL: Towards direct deposition of bioassay data. Nucleic Acids Res. 2019; 47(D1): D930–D940. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFrançois L: Elysheba/DODO: publication (v1) release. Zenodo. 2020.\n\nFrançois L: docker-ucb-public-dodo-20.04.2020 (version 20/04/2020). Zenodo. 2020." }	[ { "id": "73679", "date": "20 Nov 2020", "name": "Nicole Vasilevsky", "expertise": [ "Reviewer Expertise Mondo disease ontology", "biomedical ontology", "biocuration" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 the development of the Dictionary of Disease Ontology database that provides mappings across disease ontologies and an R package that allows users to interact with the data.\nIntroduction:\nMondo is now formatted as Mondo, not MonDO (see https://mondo.monarchinitiative.org/). You should define the abbreviations: OMIM, NCIt, etc. (Note: NCIt is formatted NCIt, not NCiT).\n\nReferences 12 and 14 are not appropriate references for the Disease Ontology.\n\nThis sentence \"While efforts such as MonDO and EFO try to integrate different disease ontologies through semantic learning and manual curation, these resources, like the different disease ontologies themselves, are currently not providing a complete mapping across all diseases ontologies\" references papers about MalaCards and BioSearch, not Mondo or EFO.\n\nMethods:\n\n\"Most mappings are unambiguous (one concept in an ontology is related to only one concept in another ontology); however, some concepts map to many similar concepts within the same ontology. This is conceptually visualized in Figure 3 where the MONDO identifier maps to 2 ORPHA identifiers.\" An important feature of Mondo is that it contains semantics with the database cross references (dbxrefs), that include precise inter-ontology relationships in the form of OWL equivalence axioms, and these relationships have the properties of symmetry and transitivity. The dbxrefs in Mondo have annotations including MONDO:equivalentTo or MONDO:relatedTo, which indicate the relationship of the referenced term to the Mondo term. If the dbxref lacks an equivalence or relatedTo axiom, it is not intended to be interpreted as such.\n\nIn your example in Figure 3, you state that Mondo maps to 2 Orphanet identifiers. There are many cases in Mondo where terms cross-reference more than one Orphanet term, but the semantics are defined. In general, Mondo does not have equivalence axioms to 2 Orphanet terms (with the rare exception of proxy merges, where we decide that two concepts in an external resource mean the same thing. In this case, we work with the source ontology (such as Orphanet) to resolve this as best as possible.) More commonly, there are dbxrefs to more than one Orphanet term where one term is equivalent, and another defined as a subClassOf or superClassOf. For example, MONDO_0011822 'Bartter disease type 3' (Orphanet:112 has the axiom annotation MONDO:subClassOf and Orphanet:93605 has the axiom annotation MONDO:equivalentTo). Further description of axiom annotation is available here: https://mondo.readthedocs.io/en/latest/editors-guide/f-entities/#axiom-annotations\n\nIn your example in Figure 4, ICD:87.0 is not an equivalent mapping to MONDO_0010561 Coffin-Lowry syndrome, and is annotated with the source Orphanet:192, meaning this mapping came from Orphanet. The semantics of the Orphanet mappings are not defined, and should not be interpreted as equivalence mappings.\n\nFigure 4: You are missing some xrefs from Mondo to other sources, see: https://www.ebi.ac.uk/ols/ontologies/mondo/terms?iri=http%3A%2F%2Fpurl.obolibrary.org%2Fobo%2FMONDO_0010561 Please see this publication on the algorithm used to develop Mondo: https://www.biorxiv.org/content/10.1101/048843v3\n\nFigure 6: The title should say ontology = Mondo (Monarch is not an ontology).\n\nUse Cases:\nUse case 2: Strict indirect conversion \"As there is no direct mapping provided by the resources between MonDO and Orphanet, transitive mapping provided by DODO needs to be applied.\" This is because Orphanet does not have a class for 'epilepsy'. ORPHA:166463 is 'epilepsy syndrome', which is mapped (MONDO:equivalentTo) to a more granular class (subclass) in Mondo: MONDO_0015650 'epilepsy syndrome'.\n\nORPHA:101993 does not return any results in Orphanet: https://www.ebi.ac.uk/ols/search?q=orphanet%3A101993&groupField=iri&start=0&ontology=ordo (or via searching here: https://www.orpha.net/consor/cgi-bin/Disease_Search_Simple.php?lng=EN). I believe this is meant to be ORPHA:101998 (not ORPHA:101993). We obsoleted this term in Mondo and merged it with MONDO:0005027, as Mondo does not include \"rare\" disease terms (see: https://github.com/monarch-initiative/mondo/issues/254).\n\n\"...the majority of MonDO identifiers cannot be converted to EFO or MeSH identifiers.\" EFO and MESH are broader than Mondo and Mondo never intended to fully map to these two terminologies. EFO does not import the entirety of Mondo, EFO only imports relevant disease terms from Mondo into their disease hierarchy that are needed for their applications.\n\nFigure 9: \"Looking into this identifier in more detail shows that it only has six direct cross-references among which one DO identifier (DOID:0050564) (Figure 9).\" Mondo only xrefs 4 sources as equivalent terms for this term, see https://www.ebi.ac.uk/ols/ontologies/mondo/terms?iri=http%3A%2F%2Fpurl.obolibrary.org%2Fobo%2FMONDO_0019587. Your figure shows relationships to 7 terms, not 6. MONDO:0019587 does not xref OMIM:618533, there is a superclassOf relationship between those two terms.\n\nFigure 10: KEGG:05014 is equivalent to MONDO:0004976 in Mondo.\n\nFigure 12: The labels are cut off, please revise this figure so the labels are displayed properly. It is my understanding that you are saying Mondo does not classify 'spinocerebellar ataxia type 2' as a child of 'familial amyotrophic lateral sclerosis' and 'familial amyotrophic lateral sclerosis' as child of 'amyotrophic lateral sclerosis', but this is not the case. This hierarchy is inferred in Mondo, I recommend you view the OWL file with the reasoner turned on and you can see this hierarchy (The Mondo OWL file is available here: https://github.com/monarch-initiative/mondo).\n\nFigure 13: The labels are cut off or missing, please review this figure so the labels are displayed properly.\n\nConclusions Is this resource currently being used by the community? Please include any description of community use and evaluation.\n\nData availability\nThe link to the docker hub returns a 404 error: https://hub.docker.com/repository/docker/elysheba/dodo\n\nI was able to access the Docker image via Zenodo, minor comments: I believe you are referring to the Mondo disease ontology, not the Monarch Ontology.\n\nHPO is not a disease ontology per se, it is an ontology of abnormal human phenotypes encountered in human diseases.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "6208", "date": "23 Dec 2020", "name": "Liesbeth François", "role": "Author Response", "response": "Thank you for providing your comments to the manuscript “Dictionary of disease ontologies (DODO): a graph database to facilitate access and interaction with disease and phenotype ontologies”, please find our replies to your comments and questions below. We would like to address your different comments and ask if you have any additional comments or thoughts. We will adapt our manuscript as described below with the receival of the second review so that we can address both reviews equally. We would also precise more generally that DODO aims to connect the information from different disease ontologies. The information from these different sources is considered equally without any priority among them. In contrast with the disease ontologies themselves that aim to harmonize and structure diseases themselves, DODO aims to facilitate the connection of different biomedical resources by relying on these different ontologies to support downstream analyses. DODO provide several parameters to flexibly obtain the most relevant identifiers for the use in mind. Introduction: Mondo is now formatted as Mondo, not MonDO (see https://mondo.monarchinitiative.org/). You should define the abbreviations: OMIM, NCIt, etc. (Note: NCIt is formatted NCIt, not NCiT). Thanks you for your comment, the abbreviations used within the document will be verified and corrected. We will also include a definitions of the abbreviations. References 12 and 14 are not appropriate references for the Disease Ontology. Thanks for you comment, we will remove these references. This sentence \"While efforts such as MonDO and EFO try to integrate different disease ontologies through semantic learning and manual curation, these resources, like the different disease ontologies themselves, are currently not providing a complete mapping across all diseases ontologies\" references papers about MalaCards and BioSearch, not Mondo or EFO. We apologize for this error, F1000 Research request weblinks and URLs to be added as a hyperlink to the manuscript (https://f1000research.com/for-authors/article-guidelines/software-tool-articles, section 13). However, during the manuscript preparation the weblinks for Mondo and EFO were removed from the manuscript by accident. We will amend this error and link to webpage of Mondo Disease Ontology (https://mondo.monarchinitiative.org/) and EFO (http://blog.opentargets.org/2019/12/19/efo3-a-community-driven-ontology-to-advance-clinical-discoveries/) which lists the resources that are currently included into these resources. The references to Malacards (Rappaport et al. 2013) and BioSearch (Hu et al. 2017) highlight more generally the issues in the completeness of mappings arising across the multitude of disease ontologies that exist. To address this potential confusion, we will change this sentence as follows: “The connections provided by existing disease ontologies are not a complete mapping across different ontologies, complicating the integration of biomedical resources (Hu et al. 2017, Rappaport et al. 2013). Similarly, ongoing efforts such as Mondo and EFO that aim to integrate different disease ontologies through semantic learning and manual curation, are currently not yet providing a complete mapping across all diseases ontologies.” Methods: \"Most mappings are unambiguous (one concept in an ontology is related to only one concept in another ontology); however, some concepts map to many similar concepts within the same ontology. This is conceptually visualized in Figure 3 where the MONDO identifier maps to 2 ORPHA identifiers.\" An important feature of Mondo is that it contains semantics with the database cross references (dbxrefs), that include precise inter-ontology relationships in the form of OWL equivalence axioms, and these relationships have the properties of symmetry and transitivity. The dbxrefs in Mondo have annotations including MONDO:equivalentTo or MONDO:relatedTo, which indicate the relationship of the referenced term to the Mondo term. If the dbxref lacks an equivalence or relatedTo axiom, it is not intended to be interpreted as such. Thanks for your clarification. This figure was created to conceptually explain the notion of ambiguity and does not capture a true example. We will adapt the figure to avoid the implication that these semantics are not considered in Mondo. Conceptually, it is sometimes possible that multiple nodes of one ontology are related through a cross-reference edge to one identifier in another ontology. The reason is that DODO uses the information provided by different ontologies which may provide additional mappings between identifiers. This is captured by the ambiguity property in the data model. In your example in Figure 3, you state that Mondo maps to 2 Orphanet identifiers. There are many cases in Mondo where terms cross-reference more than one Orphanet term, but the semantics are defined. In general, Mondo does not have equivalence axioms to 2 Orphanet terms (with the rare exception of proxy merges, where we decide that two concepts in an external resource mean the same thing. In this case, we work with the source ontology (such as Orphanet) to resolve this as best as possible.) More commonly, there are dbxrefs to more than one Orphanet term where one term is equivalent, and another defined as a subClassOf or superClassOf. For example, MONDO_0011822 'Bartter disease type 3' (Orphanet:112 has the axiom annotation MONDO:subClassOf and Orphanet:93605 has the axiom annotation MONDO:equivalentTo). Further description of axiom annotation is available here: https://mondo.readthedocs.io/en/latest/editors-guide/f-entities/#axiom-annotations Figure 3 is meant to provide a conceptual example to introduce and explain the ambiguity property and does not necessarily capture a true example. We will adapt the figure to avoid the implication that these semantics are not considered in Mondo. Additional cross-reference edges to a node may be derived from other ontologies as DODO processes and contains the information provided by different ontologies. DODO aims to facilitate access and exploration of many different disease ontologies. It doesn’t aim to curate these resources but rather build upon the extensive efforts already performed by the ontologies themselves. However, this may result in additional mappings between identifiers which is captured by the ambiguity property in the data model. In your example in Figure 4, ICD:87.0 is not an equivalent mapping to MONDO_0010561 Coffin-Lowry syndrome, and is annotated with the source Orphanet:192, meaning this mapping came from Orphanet. The semantics of the Orphanet mappings are not defined, and should not be interpreted as equivalence mappings. DODO processes multiple ontologies as a way to enrich mappings with the aim to facilitate connecting various biomedical databases. Figure 4 aimed to highlight the ambiguity derived from some mappings, in this case the ICD10 identifier Q87.0 in this disease network around Coffin-Lowry syndrome. Using transitivity mappings, the property of ambiguity is required to carefully consider which mappings to include. This ambiguity in mappings derived from different ontologies (DODO processes information from 9 different resources) and it is related to the different way disease definition are defined and the precision of the different cross-reference edges. Figure 4: You are missing some xrefs from Mondo to other sources, see: https://www.ebi.ac.uk/ols/ontologies/mondo/terms?iri=http%3A%2F%2Fpurl.obolibrary.org%2Fobo%2FMONDO_0010561 Please see this publication on the algorithm used to develop Mondo: https://www.biorxiv.org/content/10.1101/048843v3 For visibility, only a part of this disease network is shown in Figure 4. This figure aimed to introduce an example to show the issue of ambiguity of some cross-references edges (ICD10:Q87.0 in this figure) that related to a high number of mappings. We will clarify in the manuscript that the Figure 4 represent only a part of the disease network for visibility reasons. Figure 6: The title should say ontology = Mondo (Monarch is not an ontology). Thanks you for your comment, we will correct the title to represent to correct ontology name. Use Cases: Use case 2: Strict indirect conversion \"As there is no direct mapping provided by the resources between MonDO and Orphanet, transitive mapping provided by DODO needs to be applied.\" This is because Orphanet does not have a class for 'epilepsy'. ORPHA:166463 is 'epilepsy syndrome', which is mapped (MONDO:equivalentTo) to a more granular class (subclass) in Mondo: MONDO_0015650 'epilepsy syndrome'. Indeed, there is no direct cross-reference relationship as Orphanet relates to rare disorders. However, dependent on the use case, DODO allows to only use direct cross-references (to stay close to the original level) or include indirect relationships to connect various biomedical databases. These databases may not consider disease in the same way but connecting these resources may be required to perform downstream analyses. This conversion or extension is not trivial and we aimed to showcase the different possibilities within the use cases in the manuscript. Important to note is that while ontologies, such as Mondo, aim to harmonize disease definitions, DODO wants to connect the information from different ontologies with the aim to provide a flexible way to connect multiple biomedical resources that can be used for downstream analyses. There are different parameters available within the conversion and extension functions to obtain the relevant disease network or conversion that can be adapted based on the need. An example is using transitivity to connect biomedical resources that are not connected using first level cross-reference edges (different use cases for indirect conversion). However, while DODO provides a default setting, the definition of these parameters is left to the user’s requirements. ORPHA:101993 does not return any results in Orphanet: https://www.ebi.ac.uk/ols/search?q=orphanet%3A101993&groupField=iri&start=0&ontology=ordo (or via searching here: https://www.orpha.net/consor/cgi-bin/Disease_Search_Simple.php?lng=EN). I believe this is meant to be ORPHA:101998 (not ORPHA:101993). We obsoleted this term in Mondo and merged it with MONDO:0005027, as Mondo does not include \"rare\" disease terms (see: https://github.com/monarch-initiative/mondo/issues/254). Indeed, it should refer to ORPHA:101998 instead of ORPHA:101993, we will adapt this in the manuscript. The two Orphanet identifiers where not directly derived from Mondo ontology, rather they are still retained within the MedGen ontology which provides mappings between UMLS:C0014544 and ORPHA:101998 and ORPHA:166463, as can be seen in Figure 7. This example additionally shows that different ontologies provide different definitions, curation steps and rules for defining cross-reference mappings. DODO doesn’t aim to perform any curation steps on the mapping themselves, but rather build unto the extensive efforts performed by each ontology. While disease ontologies aim to provide a harmonization of disease definitions, DODO uses this information to build a more enriched disease mapping and facilitate the connection of different biomedical databases that can be used for downstream analyses. In addition, the exploration and visualization of disease networks may highlight potential errors in the original ontologies and their provided mappings. In such case, this potential error is reported to the ontology for revision. \"...the majority of MonDO identifiers cannot be converted to EFO or MeSH identifiers.\" EFO and MESH are broader than Mondo and Mondo never intended to fully map to these two terminologies. EFO does not import the entirety of Mondo, EFO only imports relevant disease terms from Mondo into their disease hierarchy that are needed for their applications. The ontologies of EFO and MeSH indeed extend beyond diseases only. However, the information processed from these resources in the scope of DODO only considers identifiers related to disease from EFO and MeSH. We will add this clarification to the manuscript, by adding the following sentence to the section discussion the feeding of the database: \"Some ontologies such as [EFO](https://www.ebi.ac.uk/efo/faq.html#whatisefo) and [MeSH](https://www.nlm.nih.gov/mesh/qualifiers_scopenotes.html) extend their ontology to include information like anatomy, disease and chemical compounds. In the scope of DODO only the relevant information on disease identifiers is extracted from these ontologies.\". However, converting all MonDO identifiers to EFO/MeSH (disease) ontology using the different possible parameters available for conversion, only a smaller subset of Mondo identifiers could be converted to a EFO/MeSH disease term. This may be a result of different ontologies that use different disease definitions, curation steps and rules for defining cross-reference mappings. Figure 9: \"Looking into this identifier in more detail shows that it only has six direct cross-references among which one DO identifier (DOID:0050564) (Figure 9).\" Mondo only xrefs 4 sources as equivalent terms for this term, see https://www.ebi.ac.uk/ols/ontologies/mondo/terms?iri=http%3A%2F%2Fpurl.obolibrary.org%2Fobo%2FMONDO_0019587. Your figure shows relationships to 7 terms, not 6. MONDO:0019587 does not xref OMIM:618533, there is a superclassOf relationship between those two terms. The Mondo ontology indeed only reports 5 cross-reference mappings (including Orphanet:90635). However, the mappings between MONDO:0019587 and OMIM:618533 is in this case provided by the EFO ontology, which adds this additional mapping for the MONDO:0019587 that they have integrated into the EFO ontology (https://www.ebi.ac.uk/ols/ontologies/efo/terms?iri=http%3A%2F%2Fpurl.obolibrary.org%2Fobo%2FMONDO_0019587). DODO processed 9 different resources equally and their provided cross-reference information. It uses this information to build a more enriched disease mapping. The primary aim is to facilitate the connection of different biomedical databases that can be used for downstream analyses. Figure 10: KEGG:05014 is equivalent to MONDO:0004976 in Mondo. Thanks for your comment, it is possible that you refer to Figure 11 instead? This disease network indeed shows the cross-reference mappings between MONDO:0004976 and KEGG:05014. In addition to the ambiguity property, two types of cross-reference edges are defined: is_xref and is_related. Briefly, an is_xref edges is used to define equal cross-reference relationships between ontologies are more or less similar in concept definition. The exploration of this threshold is based on 14 different ontologies that are frequently used and trusted by the authors. This type of cross-reference edge is used in obtaining all transitive mappings. An is_related edges is used for all other cross-reference edges and this relationship is not used in transitivity mappings directly. Rather, it is only used in the final step of conversion after transitivity mapping to obtain the direct relationships of the converted nodes or when applying the direct conversion (use case 1). As KEGG ontology is not considered when exploring the is_xref threshold setting, all cross-reference relationships which consider a KEGG identifier, are classified as is_related edges. Figure 12: The labels are cut off, please revise this figure so the labels are displayed properly. It is my understanding that you are saying Mondo does not classify 'spinocerebellar ataxia type 2' as a child of 'familial amyotrophic lateral sclerosis' and 'familial amyotrophic lateral sclerosis' as child of 'amyotrophic lateral sclerosis', but this is not the case. This hierarchy is inferred in Mondo, I recommend you view the OWL file with the reasoner turned on and you can see this hierarchy (The Mondo OWL file is available here: https://github.com/monarch-initiative/mondo). Thanks for your remark, we will revise the figure to display the full label. We recognize that the explanation in the manuscript can be confusing as indeed “spinocerebellar ataxia type 2” (SCA2) is classified as a child of “familial amyotrophic lateral sclerosis” which in term is a child of “amyotrophic lateral sclerosis”. This relationship in is indeed reported in EFO. As we focus on integrating information from ClinVar and CHEMBL, we only consider the relevant identifiers in this context. While this relationship is reported in Mondo, this ontology is not used to integrate the information from CHEMBL and ClinVar. We will amend the sentence to better clarify our meaning to “However, while EFO reports ‘familial amyotrophic lateral sclerosis’ as a parent term, it is unclear whether this can be considered as ALS disease clinically”. Figure 13: The labels are cut off or missing, please review this figure so the labels are displayed properly. Thanks for your remark, we will revise the figure to display the full label. Conclusions Is this resource currently being used by the community? Please include any description of community use and evaluation. This publication coincided with the first release of the database and R package for the community. We hope that the open access sharing of resource will allow and facilitate access and usage of the disease ontology resources by the community. As of yet, we have not seen any publications referring to the usage of DODO by the community as DODO has only recently been released for public usage. However, DODO is being used internally to integrating different biomedical resources and with this publication we wanted to take the opportunity to share our work with the community. Data availability The link to the docker hub returns a 404 error: https://hub.docker.com/repository/docker/elysheba/dodo We apologize for the incorrect link, the link to docker hub was corrected (https://hub.docker.com/repository/docker/elysheba/public-dodo) in the manuscript. I was able to access the Docker image via Zenodo, minor comments: I believe you are referring to the Mondo disease ontology, not the Monarch Ontology. Thanks for your comment, we will correct the ontology name to Mondo disease ontology in both the docker hub (https://hub.docker.com/repository/docker/elysheba/public-dodo) and zenodo archive (10.5281/zenodo.3921874) HPO is not a disease ontology per se, it is an ontology of abnormal human phenotypes encountered in human diseases. Indeed, we have specified this distinction more clearly in both the docker hub repository and the Zenodo archive that HPO refers to a phenotype ontology and is not a disease ontology as such." } ] }, { "id": "152033", "date": "14 Oct 2022", "name": "Jeremy G. Frey", "expertise": [ "Reviewer Expertise Digital chemistry", "Semnatic Web" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nWe like the paper from a technical perspective. The main feedback would be that it lacks some of the real world application and needs a bit more explanation around that.\nThe paper gives you a lot of info about the system technically does but doesn’t really explain why this will be useful in practice.\nOverall this is an interesting well written paper, with some well documented data and code sources. I would suggest accept with minor revisions which would add some additional value.\n\nAs this is a cross domain paper, it is particularly important to make it accessible to researchers from these different domains. As such, it would be useful to include a simple explanation of how transitive mappings work. It would also be interesting to either see an example of a similar approach to this paper, or if this does not exist state that, and explain how and why you took this approach.\n\nThis paper goes into a lot of good technical detail about how DODO was created, but it could use a bit more of an explanation about the why earlier on in the paper. You've covered this from a technical perspective, in that there is no software available to explore these ontologies as a whole, this would create a more flexible landscape etc but haven't noted why this is beneficial outside of this technical remit. What could a researcher potentially do with your tool that they couldn't do before? How will this benefit the disease community from a real world impact scenario? You mention this briefly right at the end in the conclusion, but it would be good to see these motivations stated up front.\n\nOn a similar note, I like that you have included use cases to demonstrate how researchers would use your application. However, it would be useful to see these linked back to more specific real world examples. If you convert disease and phenotype identifiers between ontologies WHY is this useful? HOW will this enable researchers to develop more novel treatments in a way that they couldn't do before?\nThere are a few areas that need some minor grammatical changes / tense changes\nDODO combines the information provided by the different ontologies and allows connecting ontologies to one another, even if they don’t have direct cross-references — weird sentence, would suggest changing allows to enables?\n\nStructuring and harmonize the information derived from each ontology - changed tense within the sentence. Structure and harmonize.\n\nThis is exemplified in Figure 3, where transitivity mappings is needed to connect the initial MONDO identifier - mappings ARE needed.\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": [] } ]	1	https://f1000research.com/articles/9-942
https://f1000research.com/articles/9-941/v1	07 Aug 20	{ "type": "Study Protocol", "title": "ACCEPT - combining acalabrutinib with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP) for Diffuse Large B-cell Lymphoma (DLBCL): study protocol for a Phase Ib/II open-label non-randomised clinical trial", "authors": [ "Andrew Davies", "Sharon Barrans", "Cathy Burton", "Katy Mercer", "Joshua Caddy", "Fay Chinnery", "Laura Day", "Diana Fernando", "Kirit Ardeshna", "Graham Collins", "John Radford", "Simon Rule", "Andrew McMillan", "Peter Johnson", "Gareth Griffiths", "Andrew Davies", "Sharon Barrans", "Cathy Burton", "Joshua Caddy", "Fay Chinnery", "Laura Day", "Diana Fernando", "Kirit Ardeshna", "Graham Collins", "John Radford", "Simon Rule", "Andrew McMillan", "Peter Johnson", "Gareth Griffiths" ], "abstract": "Background: Over 13,000 new cases of non-Hodgkin’s lymphoma (NHL) are diagnosed in the UK, with approximately 4,900 attributable deaths each year. Diffuse Large B-cell Lymphoma (DLBCL) is the most common NHL comprising one third of adult NHL cases. R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone) is accepted as the international standard first-line regimen, but improvement in first line treatment is needed. Dysregulated B-cell receptor (BCR) signalling has been identified as a feature of DLBCL. Inhibition of Bruton’s tyrosine kinase (Btk), downstream of the BCR has proven efficacious in other B-cell malignancies and in combination with R-CHOP. The second generation Btk inhibitor, acalabrutinib, may have improved target potency and specificity, and therefore better efficacy and tolerability. Methods: ACCEPT is an open-label non-randomised Phase Ib/II trial testing the addition of acalabrutinib to conventional R-CHOP therapy. ACCEPT incorporates an initial 6+6 modified Phase I design of up to 24 participants followed by 15 participant single arm Phase II expansion cohort in treatment naive patients with histologically confirmed DLBCL expressing CD20. Participants are recruited from UK secondary care sites. Phase I will establish the recommended Phase II dose (RP2D, primary endpoint) of acalabrutinib in combination with R-CHOP. Phase II will gain additional information on safety and efficacy on the RP2D. The primary endpoints of Phase II are overall response rate and toxicity profile. Secondary endpoints include duration of response (progression-free survival and overall survival OS) in relation to cell of origin. Analyses are not powered for formal statistical comparisons; descriptive statistics will describe rates of toxicity, efficacy and translational endpoints. Discussion: ACCEPT will provide evidence for whether acalabrutinib in combination with R-CHOP is safe and biologically effective prior to future Phase II/III trials in patients with previously untreated CD20 positive DLBCL. Trial registration: EudraCT Number: 2015-003213-18 (issued 16 July 2015); ISRCTN13626902 (registered 07 March 2017).", "keywords": [ "Diffuse large B-cell lymphoma", "acalabrutinib", "Btk inhibitor", "R-CHOP", "molecular profiling", "phase I/II" ], "content": "Abbreviations\n\nABC, Activated B-cell; ADCC, Antibody dependent cellular cytotoxicity; AE, Adverse Event; AUC, Area under curve; BCR, B-cell Receptor; Btk, Bruton Tyrosine Kinase; CHOP, Cyclophosphamide, doxorubicin, vincristine, prednisolone; CLL, Chronic lymphocytic Leukaemia; Cmax, Maximum Concentration; COO, Cell of origin; CRF, Case Report Form; CT, Computer Tomography; CTCAE, Common Terminology Criteria for Adverse Events; DMEC, Data Monitoring and Ethics Committee; DLT, Dose Limiting Toxicity; DLBCL, Diffuse Large B-cell lymphoma; ECOG, Eastern Cooperative Group; FDA, Food and Drug Administration; FFPE, Formalin Fixed Paraffin Embedded; FISH, Fluorescence In Situ Hybridization; GCB, Germinal Centre B-cell; HBcAB, Hepatitis B core Antibody; HBsAB, Hepatitis B Surface antibody; HBsAg, Hepatitis B surface Antigen; HBV, Hepatitis B virus; HCV, Hepatitis C virus; HIV, Human Immunodeficiency Virus; HMDS, Haematological Malignancy Diagnostic Service; IMP, Investigational Medicinal Product; IV, Intravenous; LVEF, Left Ventricular Ejection Fraction; MAD, Maximal administered Dose; MHRA, Medicines and Healthcare products Regulatory Agency; MTD, Maximum Tolerated Dose; NCI, National Cancer Institute; NHL, Non-Hodgkin Lymphoma; Od, Once daily; ORR, Overall Response Rate; PBMC, Peripheral Blood Mononuclear cells; PK, Pharmacokinetic; PD, Progressive disease; PET CT, Positron Emission Tomography computer tomography; PIS, Patient Information Sheet; PO, By mouth; PR, Partial Response; RP2D, Recommended Phase 2 Dose; R-CHOP, Rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone; SAE, Serious Adverse Event; SCTU, Southampton Clinical Trial Unit; SUSAR, Suspected Unexpected Serious Adverse Reaction; Tmax, Time to maximum concentration\n\n\nIntroduction\n\nMore than 13,000 new cases of non-Hodgkin’s lymphoma (NHL) were diagnosed in the UK between 2014–2016, with nearly 4,900 patients dying as a result of their disease in 20171. Diffuse large B-cell lymphoma (DLBCL) is the most common type of NHL, accounting for 30–40% of NHL cases in adults with an annual incidence of about 5,500 patients in the UK1,2.\n\nCyclophosphamide, vincristine, doxorubicin and prednisolone (CHOP) used to be the standard of care for DLBCL. But, overall survival was disappointing; CHOP only cured about 30 percent of patients with advanced stages of intermediate-grade or high-grade NHL3. Addition of the anti-CD20 monoclonal antibody rituximab to CHOP resulted in higher response rates, longer event free survival and improved overall survival. R-CHOP is now accepted as the international standard, with cure rates around 75%4–8.\n\nDespite this improvement, many patients either fail to respond or relapse after having achieved an initial remission. Their prognosis is poor, because earlier rituximab therapy limits the success of salvage treatment and so the majority of these patients will die from their disease9. Consequently, there is an urgent need to improve the effectiveness of first line treatment.\n\nGene expression profiling of untreated DLBCL samples has identified three distinct sub-classifications of the disease: activated B-cell like [ABC-DLBCL]; germinal centre B-cell like [GCB- DLBCL] and unclassified DLBCL. Each has its own biological features and clinical outcomes when treated with CHOP or R-CHOP.\n\nThe cells of ABC type are driven by “chronic active” B-cell receptor (BCR) signalling10. The BCR signalling pathway can be disrupted using Bruton tyrosine kinase (Btk) inhibitors. Targeting BCR signalling by inhibiting Btk with ibrutinib (a first generation Btk inhibitor) in combination with R-CHOP is known to be safe for previously untreated B-cell NHL11.\n\nAcalabrutinib (Acerta Pharma B.V.) is a second generation Btk inhibitor with increased target selectivity compared to ibrutinib12. As of 30 December 2018, acalabrutinib has been administered to over 2600 participants in clinical studies, including patients with haematologic malignancies, solid tumour, or rheumatoid arthritis, and participants who are healthy volunteers or with mild to moderate hepatic impairment. No serious adverse events (SAEs) have been reported in the hepatic impairment study or in the healthy volunteer studies. No expected SAEs have been identified for acalabrutinib to date.\n\nWe hypothesised that acalabrutinib would have improved target potency and specificity and is both active and tolerable in combination with R-CHOP.\n\n\nObjectives\n\nThe objectives of the study are described in Table 1. ACCEPT aims to establish a recommended Phase II dose (RP2D) for acalabrutinib when combined with R-CHOP.\n\nSecondary objectives include the evaluation of dose limiting toxicity (DLT), assessed using CTCAE v4.03 at baseline, at each treatment cycle and at each follow-up visit; treatment compliance, assessed using patient diaries and returned Investigational Medicinal Product (IMP), as well as the electronic case report forms (eCRFs) during the treatment period; and response rate of participants enrolled in the Phase II part of the trial.\n\nTranslational objectives include determining the pharmacokinetic (PK) profile of acalabrutinib when given in combination with R-CHOP in patients with DLBCL. Btk occupancy by acalabrutinib of PBMC peripheral blood mononuclear cells (PBMCs) will be measured using fluorescent affinity probe assay. Pre- and post-dose blood samples will be used to measure the impact of the addition of acalabrutinib on R-CHOP mediated antibody dependent cellular cytotoxicity (ADCC), and measure BCR activation via CD86 and CD69 expression on PBMC by flow cytometry.\n\nACCEPT will prospectively validate the cell of origin (ABC versus GCB) model of DLBCL and its practicality and utility, as well as assessing the benefit/toxicity of the addition of acalabrutinib to R-CHOP. DNA extracted from tumour material will be used to perform mutation detection on BCR pathway (e.g. Btk, PI3K, CD79b). Genetic abnormalities of BCR pathway will be correlated with clinical outcomes and the expression of BCR pathway target genes.\n\nACCEPT is a multicentre open-label non-randomised Phase Ib/II clinical trial conducted in two stages recruiting approximately 40 participants. Phase I will be dose escalation following the conventional rules of 6+6 modified design, escalation will proceed until a maximum tolerated dose (MTD) is defined or the maximal administered dose (MAD) is determined in order to define the RP2D. A six participant cohort design is employed to maximise patient safety13, which will result in 6–24 participants recruited. Phase II will be an expansion cohort in order to gain additional information on safety and efficacy at the RP2D from a total of 15 participants recruited (Figure 1).\n\n\nMethods: participants, interventions and outcomes\n\nThis is protocol V6 (15-Jul-2019).\n\nSeven secondary care hospitals in the UK are recruiting participants into ACCEPT. A list of study sites is available via contacting accept@soton.ac.uk.\n\nInclusion criteria\n\nParticipants should fulfil the following criteria:\n\nHistologically confirmed DLBCL, expressing CD20; sufficient tumour block should be available to forward to a central laboratory for gene expression profiling and pathology review; at a minimum, participants should have sufficient tumour material to test for: H/E morphological check of compatibility of DLBCL diagnosis, immunophenotyping and RNA for gene expression profiling\n\nMeasurable disease of at least 15mm\n\nNot previously treated for lymphoma and fit enough to receive combination chemoimmunotherapy with curative intent\n\nStage IAX (bulk defined as lymph node diameter >10cm) to stage IV disease and deemed to require a full course of chemotherapy. Patients with non-bulky IE disease will not be eligible\n\nECOG performance status 0–2 or 3 if this is directly attributable to lymphoma\n\nAdequate bone marrow function with platelets > 100×109/L; neutrophils > 1.0×109/L at study entry, unless lower figures are attributable to lymphoma\n\nMeasured or calculated creatinine clearance > 30mls/min, (calculated using the formula of Cockcroft and Gault [(140-Age) × Mass (kg) × (1.04 (for women) or 1.23 (for men))/Serum Creatinine (µmolL)]\n\nSerum bilirubin < 35μmol/L and transaminases < 2.5× upper limit of normal at time of study entry\n\nCardiac function sufficient to tolerate 300mg/m2 of doxorubicin. A pre-treatment echocardiogram or MUGA is required to establish baseline LVEF equal to or greater than institutional normal range.\n\nNo concurrent uncontrolled medical condition\n\nLife expectancy > 3 months\n\nAged 16 years and above\n\nWilling and able to participate in all required evaluations and procedures in this study protocol including swallowing capsules without difficulty\n\nAbility to understand the purpose and risks of the study and provide signed and dated informed consent\n\nExclusion criteria\n\nPatients will be excluded from the study entry if any of the following criteria are met:\n\nPrevious history of treated or untreated indolent lymphoma; however newly diagnosed patients with DLBCL who are found to also have small cell infiltration of the bone marrow or other diagnostic material (discordant lymphoma) will be eligible\n\nPatients who have received immunisation with a live vaccine within four weeks prior to enrolment will be ineligible\n\nDiagnosis of primary mediastinal lymphoma\n\nDiagnosis of primary Central Nervous System lymphoma\n\nHistory of stroke or intracranial haemorrhage in preceding 6 months\n\nHistory of bleeding diathesis (e.g., haemophilia, von Willebrand disease)\n\nRequires or receiving anticoagulation with warfarin or equivalent antagonists (e.g. phenprocoumon) within 7 days of first dose of acalabrutinib; however, patients using therapeutic low molecule weight heparin or low dose aspirin will be eligible\n\nPrior exposure to a BCR inhibitor (e.g. Btk inhibitors, phosphoinositide-3 kinase (PI3K), or Syk inhibitors) or BCL-2 inhibitor (e.g. ABT-199)\n\nRequires treatment with a strong cytochrome P450 3A4 (CYP3A4) inhibitor/inducer.\n\nRequires treatment with proton pump inhibitors (e.g. omeprazole, esomeprazole, lansoprazole, dexlansoprazole, rabeprazole, or pantoprazole)\n\n○ Patients receiving proton pump inhibitors should switch to short-acting H2-receptor antagonists or antacids prior to study entry to be eligible for enrolment into this study\n\nUncontrolled systemic infection.\n\nMajor surgery in the preceding 4 weeks of first dose of study drug. If a subject had major surgery, they must have recovered adequately from any toxicity and/or complications from the intervention before the first dose of study drug\n\nSignificant cardiovascular disease such as uncontrolled or symptomatic arrhythmias, congestive heart failure, or myocardial infarction within 6 months of screening, or any Class 3 or 4 cardiac disease as defined by the New York Heart Association Functional Classification, or corrected QT interval (QTc) > 480 msec at screening. QTc interval should be calculated using Fridericia’s formula\n\nSerological positivity for Hepatitis B (HBV), C (HCV), or known HIV infection. As per standard of care, prior to initiation of immunochemotherapy, the results of hepatitis serology should be known prior to commencement of therapy.\n\n- Positive test results for chronic HBV infection (defined as positive HBsAg serology) will not be eligible. Patients with occult or prior HBV infection (defined as negative HBsAg and positive total HBcAb) will not be eligible. Patients who have protective titres of hepatitis B surface antibody (HBsAb) after vaccination will be eligible\n\n- Positive test results for HCV (HCV antibody serology testing) will not be eligible\n\nWomen who can bear children must agree to use two highly effective forms of contraception or abstinence during the study and for 12 months after the last treatment dose (contraception is discussed under ‘5. Relevant concomitant care permitted or prohibited during the trial’)\n\nBreastfeeding or pregnant women\n\nMen who can father children must agree to use two highly effective forms of contraception with additional barrier or abstinence during the study and for 12 months after the last treatment dose (contraception is discussed under ‘Relevant concomitant care permitted or prohibited during the trial’)\n\nMen must agree to refrain from sperm donation during the study and for 12 months after the last treatment dose\n\nSerious medical or psychiatric illness likely to affect participation or that may compromise the ability to give informed consent\n\nPrior malignancy (other than DLBCL), except for adequately treated basal cell or squamous cell skin cancer, in situ cervical cancer, or other cancer from which the subject has been disease free for ≥ 2 years or which will not limit survival to < 2 years. Note: these cases must be discussed with Southampton Clinical Trials Unit (SCTU)\n\nMalabsorption syndrome, disease significantly affecting gastrointestinal function, resection of the stomach or small bowel, gastric bypass, symptomatic inflammatory bowel disease, or partial or complete bowel obstruction or gastric restrictions and bariatric surgery, such as gastric bypass\n\nAny immunotherapy within 4 weeks of 1st dose of the study\n\nConcurrent participation in another therapeutic clinical trial\n\nWho will take informed consent?\n\nConsent to enter the trial will be sought from each participant only after a full explanation has been given, a Patient Information Sheet (PIS) offered (Extended data14) and time allowed for consideration. Signed participant consent will be obtained. Only site staff named on the Delegation Log and authorised to do so may obtain consent. Patients may refuse to participate without giving reasons and this will not prejudice their future treatment.\n\nAdditional consent provisions for collection and use of participant data and biological specimens\n\nThe trial’s Informed Consent Forms detail the consent provisions for collection and use of participant data and biological specimens in future research (Extended data14).\n\nExplanation for the choice of comparators\n\nRationale for combination of acalabrutinib in combination with R-CHOP in DLBCL\n\nDysregulation of BCR signalling is well recognised in DLBCL and other B-cell malignancies. Btk is central to signalling through the BCR. The safety and efficacy of a first generation Btk inhibitor, ibrutinib, in combination with R-CHOP has already been investigated by a Phase Ib study in patients with DLBCL, demonstrating that it is well tolerated with an overall response rate (ORR) of 91% (CR 70%)10.\n\nAcalabrutinib is a second generation Btk inhibitor with enhanced kinase selectivity and potential for better efficacy and tolerability over first-generation inhibitors. It has a number of preclinical properties that would indicate the potential for favourable efficacy and safety over ibrutinib. These include preferential selectivity for Btk with the potential for less off-target effects (e.g. Epidermal Growth Factor Receptor and diarrhoea). Acalabrutinib does not appear to abrogate thrombus formation, compared to ibrutinib, mediated by off-target kinase activity, and would therefore potentially reduce the risk of bleeding and also compared to ibrutinib does not have a negative effect on ADCC, an important mediator of rituximab function.\n\nResults from the study of acalabrutinib in patients with chronic lymphocytic leukaemia (CLL) demonstrate a favourable toxicity profile with an emergent lower rate of haematological toxicity compared to ibrutinib. No DLTs were reached and no SAEs reported to date at doses ≤400mg15.\n\nRationale for dose selection\n\nResults from the study of ibrutinib in combination with R-CHOP indicated that the maximal tolerated dose was not reached and the recommended Phase II dose of ibrutinib was 560mg od10. This is the licensed single agent dose for use in mantle cell lymphoma. Given the favourable single agent toxicity profile of acalabrutinib in the CLL study compared to ibrutinib, it is anticipated that acalabrutinib may safely be given in combination with R-CHOP at doses close or equivalent to those being investigated as a single agent.\n\nPreliminary data from the ongoing Phase I/II study in patients with relapsed/refractory or previously untreated CLL have shown that acalabrutinib is well tolerated at dosages of 100 to 400 mg od and 100 to 200 mg bd15. These data suggest that de novo synthesis of Btk can occur within 24 hours in peripheral blood cells. Twice daily dosing may ensure Btk inhibition for the entire 24 hours and thus may be beneficial in terms of increasing efficacy and/or decreasing development of resistance to acalabrutinib.\n\nTaken together, the proposed starting dose of 100mg od is considered represent one where there is good pharmacodynamic evidence that the target is being suitably occupied without safety concerns in single agent studies and one supported by our pharmacokinetic knowledge of acalabrutinib. Escalation to twice daily dosing in the second cohort, addresses the continued inhibition of Btk based upon concerns about de novo synthesis during the 24-hour period.\n\nIntervention description\n\nAll participants will receive 6 cycles of R-CHOP on a standard 21 day schedule with the addition of acalabrutinib in cycles 2–6. This will be followed by a continuation period of acalabrutinib only for 2 cycles each of 28 days. The Schedule of Events (Table 2) describes the trial interventions.\n\na Screening investigations to be performed within 14 days of starting study medication with the exception of informed consent, CT, Electrocardiogram and Bone marrow biopsy\n\nb Blood pressure, pulse, temperature, height and weight. Assessment to be performed pre-dose and as per local practice during rituximab infusion\n\nc PET and Contrast Enhanced CT of chest, abdomen and pelvis (neck if indicated) should be carried out within 35 days of planned treatment. The PET-CT hybrid scanners may be used to acquire the required CT images only if the CT produced by the scanner is of diagnostic quality and includes the use of intravenous (IV) contrast. If this cannot be achieved, a PET and separate Contrast Enhanced CT scan should be performed. Bi-dimensional measurements are expected.\n\nd Bone marrow aspirate and trephine biopsy (single site with adequate trephine) (within 90 days of first treatment).\n\ne Serum chemistry to include sodium, potassium, urea, creatinine, bilirubin, alanine or aspartate transaminase, alkaline phosphatase and albumin.\n\nf Additional serum chemistry to be performed only at baseline: LDH, calcium, phosphate, β2-microglobulin and uric acid.\n\ng Full blood count to include haemoglobin, white blood cell count, absolute neutrophil, lymphocytes and platelets counts. To be taken within 72 hours of chemotherapy administration on each cycle\n\nh As per standard of care, sites should have hepatitis results available prior to initiation of immunochemotherapy.\n\ni Only required in females of child bearing potential on day 1 before treatment commences\n\nj A 12 lead ECG should be performed on all patients.\n\nk In addition, an Echocardiogram or MUGA will be performed for all patients to establish a left ventricular ejection fraction equal to or greater than institutional normal range. Patients should be considered suitable to receive 300mg/m2 doxorubicin\n\nl Cerebrospinal fluid examination should be performed if clinically indicated or lymphomatous involvement of peripheral blood, nasal/paranasal sinuses or testis. CNS prophylaxis may be given according to local policy\n\nm Diagnostic tumour block to be forwarded immediately upon obtaining either Tissue Block Screening consent or main study consent (for those patients whose tissue sample is easily accessible) to HMDS, Leeds, according to study procedure outlined in Investigator Site File.\n\nn 20ml Blood sample in 2x10ml Streck tubes and 1x7.5ml EDTA Blood sample to be forwarded immediately to HMDS, Leeds prior to treatment start.\n\no Adverse Events to be collected from date of consent. Only Adverse Events related to study procedures should be reported prior to start of treatment.\n\np Blood pressure, pulse and temperature. Assessment to be performed pre-dose and as per local practice during rituximab infusion\n\nq PET and Contrast Enhanced CT of chest, abdomen and pelvis (neck if indicated) needs to be completed within 3 weeks of the patient completing last treatment cycle. The PET-CT hybrid scanners may be used to acquire the required CT images only if the CT produced by the scanner is of diagnostic quality and includes the use of intravenous (IV) contrast. If this cannot be achieved, a PET and separate Contrast Enhanced CT scan should be performed. Bi-dimensional measurements are expected.\n\nr Bone marrow biopsy to be repeated at the end of treatment if initially involved (to confirm CR)\n\ns Serum chemistry to include sodium, potassium, urea, creatinine, bilirubin, alanine or aspartate transaminase, alkaline phosphatase, LDH and albumin. To be taken within 72 hours of administration of each cycle\n\nt Full blood count including haemoglobin, white cell count, absolute neutrophil count, lymphocytes and platelets. To be taken within 72 hours of chemotherapy administration on each cycle\n\nu Compliance will be assessed by patient’s diary with date and time of acalabrutinib administration, and the count of remaining capsules\n\nv 20ml Blood sample in 2x10ml Streck tubes to be forwarded immediately to HMDS, Leeds\n\nw End of RCHOP + acalabrutinib treatment at week 19 is also the start of the continuation phase of acalabrutinib. The dosing of acalabrutinib is continued after the last day of treatment (cycle 6 day 21) for 56 days further.\n\nx Acalabrutinib only is given for cycles 7 and 8 as a continuation phase on a 28 days per cycle schedule - a total of 56 days continuation phase.\n\ny Physical exam and ECOG performance status. To be taken within 48 hours of acalabrutinib administration.\n\nz Electrocardiogram and Echocardiogram to be taken within 48 hours of acalabrutinib administration.\n\naaCT scan to be completed during Week 19 (+/- 1 week)\n\nbb. EoT visit should take place within 3 weeks from the final dose of acalabrutinib.\n\ncc Post dose samples should be taken at the protocol specified time ± 5 minutes.\n\ndd Serum chemistry to include sodium, potassium, urea, creatinine, bilirubin, alanine or aspartate transaminase, alkaline phosphatase, LDH and albumin.\n\nee A contrast enhanced CT scan of the neck, chest, abdomen and pelvis will be performed at 12 months and 24 months following completion of protocol specified therapy. Bi-dimensional measurements are expected.\n\nff 20 ml Blood sample in 2x10ml Streck tubes to be forwarded immediately to HMDS, Leeds.\n\ngg A visit window of +/- 2 weeks is permitted for follow-up visits.\n\nNB: The Participant/legal representative is free to withdraw consent at any time without providing a reason. When withdrawn, the participant will continue to receive standard clinical care. Follow up data will continue to be collected (unless the participant/legal representative has specifically stated that they do not want this to happen.\n\nPhase I - Dose Escalation\n\nStage 1:\n\nR-CHOP + Acalabrutinib (Cycle 2–6)\n\n- Rituximab 375mg/m2 IV, on day 1\n\n- Cyclophosphamide 750mg/m2 IV, on day 1\n\n- Doxorubicin 50mg/m2 IV, on day 1\n\n- Vincristine 1.4mg/m2 (max 2mg) IV, on day 1\n\n- Prednisolone 100mg po, on day 1–5\n\n- Acalabrutinib, 100mg once daily taken orally, days 1–21\n\nCycle 7 (28 days) and 8 (28 days) - Acalabrutinib only\n\n- Acalabrutinib, 100mg once daily taken orally for 56 days\n\nAcalabrutinib dosage:\n\nThe first six participants enrolled in the study (cohort 1) will receive 100mg od started on day 1 of the 2nd cycle of R-CHOP. When participants of the first cohort have completed their third cycle, and their safety data has been reviewed by the Safety Review Committee, the decision for dose escalation to 100mg bd for cohort 2 could be taken.\n\nStage 2:\n\nR-CHOP + Acalabrutinib (Cycle 2–6)\n\n- Rituximab 375mg/m2 IV, on day 1\n\n- Cyclophosphamide 750mg/m2 IV, on day 1\n\n- Doxorubicin 50mg/m2 IV, on day 1\n\n- Vincristine 1.4mg/m2 (max 2mg) IV, on day 1\n\n- Prednisolone 100mg po, on day1–5\n\n- Acalabrutinib 100mg twice daily, orally days 1–21\n\nCycle 7 (28 days) and 8 (28 days) - Acalabrutinib only\n\n- Acalabrutinib, 100mg twice daily taken orally for 56 days following end of treatment with 6 cycles R-CHOP.\n\nPhase II – Dose Expansion\n\nR-CHOP + Acalabrutinib (Cycle 2–6)\n\n- Rituximab 375mg/m2 IV, on day 1\n\n- Cyclophosphamide 750mg/m2 IV, on day 1\n\n- Doxorubicin 50mg/m2 IV, on day 1\n\n- Vincristine 1.4mg/m2 (max 2mg) IV, on day 1\n\n- Prednisolone 100mg po, on day 1–5\n\n- Acalabrutinib at a Recommended Phase II dose\n\nCycle 7 (28 days) and 8 (28 days) - Acalabrutinib only\n\nAcalabrutinib, at RP2D taken orally for 56 days following end of treatment with 6 cycles R-CHOP.\n\nCriteria for discontinuing or modifying allocated interventions\n\nAfter the participant has entered the trial, the clinician remains free to give alternative treatment to that specified in the protocol at any stage if he/she feels it is in the participant’s best interest, but the reasons for doing so should be recorded. In these cases, participants will be withdrawn from protocol treatment but remain within the trial for the purposes of follow-up and data analysis. All participants are free to withdraw at any time from the protocol treatment without giving reasons and without prejudicing further treatment.\n\nStrategies to improve adherence to interventions\n\nThe investigator and/or study personnel will assess participant compliance with acalabrutinib at each study visit using direct questioning, examination of participants’ drug administration diaries and pill counts.\n\nSupportive care\n\nLive vaccination whilst on rituximab is prohibited and for participants who have peripheral B cell depletion must not have a live vaccination within three months of last rituximab dose\n\nParticipants will receive standard anti-emetic as per local policy\n\nAllopurinol 300mg orally od should be given as per local practice; rasburicase may be administered if high risk of tumour lysis syndrome\n\nMouth care therapies are given according to local policy\n\nAntacids and calcium supplements should be avoided for a period of at least 2 hours before and after taking acalabrutinib; short acting H2 receptor antagonists should only be taken at least 2 hours following acalabrutinib administration\n\nDiarrhoea and constipation are both recognised side effects of therapy in this protocol, diarrhoea is the most common; treatment should be given according to local policy, although prophylactic anti-diarrhoeal agents are not recommended\n\nGranulocyte-colony stimulating factor (GCSF) is mandated for all participants receiving R-CHOP and acalabrutinib; the formulation and duration will be according to local policy for primary prophylaxis but should be for at least seven days or until neutrophil recovery for those participants receiving the non-pegylated formulation\n\nAntimicrobial prophylaxis given against Pneumocystis jirovecii pneumonia is mandated\n\nSuitable infective prophylaxis to be given to participant’s aged 65 and over as per local policy; ciprofloxacin prophylaxis should be avoided due to possible interactions with acalabrutinib\n\nProhibited therapies\n\nAcalabrutinib is not a strong direct inhibitor or inducer or CYP isoforms; thus, acalabrutinib, at the currently used clinical doses, is unlikely to be a perpetrator of a drug interaction at the level of inhibition or induction of CYP isoforms. Acalabrutinib is partially metabolised by CYP3A; its exposure is affected when co-administered with strong CYP3A4 inducers or inhibitors. Consequently, the concomitant use of strong inhibitors/inducers of CYP3A4 (see Table 3) should be avoided when possible.\n\na. A strong inhibitor for CYP3A is defined as an inhibitor that increases the AUC of a substrate for CYP3A by ≥ 5-fold.\n\nb. In vivo inhibitor of P-glycoprotein.\n\nc. The effect of grapefruit juice varies widely among brands and is concentration-, dose-, and preparation dependent. Studies have shown that it can be classified as a “strong CYP3A inhibitor” when a certain preparation was used (eg, high dose, double strength) or as a “moderate CYP3A inhibitor” when another preparation was used (eg, low dose, single strength).\n\nd. Withdrawn from the United States market because of safety reasons.\n\ne. A strong inducer for CYP3A is defined as an inducer that results in ≥ 80% decrease in the AUC of a substrate for CYP3A.\n\nf. In vivo inducer of P-glycoprotein.\n\nNote: The list of drugs in these tables is not exhaustive. Any questions about drugs not on this list should be addressed to the SCTU\n\nSource: FDA Drug Development and Drug Interactions: Table of Substrates, Inhibitors and Inducers. Web link Accessed 21 January 2015:\n\nhttp://www.fda.gov/Drugs/DevelopmentApprovalProcess/DevelopmentResources/DrugInteractionsLabeling/ucm093664.htm#inVivo\n\nBased on these considerations, patients who require therapy with drugs listed in Table 3 should not be enrolled into the study. If medically justified, patients may be enrolled if such inhibitors or inducers can be discontinued or alternative drugs that do not affect these enzymes can be substituted within 7 days before first dose of study drug. If a subject requires a strong CYP3A4 while on study, the subject will be monitored closely for potential drug-related toxicities.\n\nThe effect of agents that reduce gastric acidity (e.g., proton pump inhibitors, H2 receptor antagonists or antacids) on acalabrutinib absorption was evaluated in a healthy volunteer study (ACE-HV-004; Acalabrutinib Investigator Brochure v8 ACERTA PHARMA B.V.). Results from this study indicate that participants should avoid the use of calcium carbonate containing drugs or supplements (e.g., antacids and calcium supplements) for a period of at least 2 hours before and after taking acalabrutinib. Similarly, participants should avoid the use of H2-receptor antagonists for a period of 2 hours after taking the study drugs. Use of omeprazole, esomeprazole, lansoprazole or any other proton pump inhibitors while taking acalabrutinib is not recommended due to a potential decrease in study drug exposure. However, if a subject requires the use of a proton pump inhibitor while on study (e.g., to treat a gastric ulcer) treatment options will be discussed with SCTU.\n\nIn circumstances where treatment with ciprofloxacin is needed, the dose of acalabrutinib should be reduced to 100mg od.\n\nWarfarin and equivalent Vitamin K antagonists are prohibited. However, participants may use therapeutic low molecule weight heparin or low dose aspirin.\n\nDietary restrictions\n\nAcalabrutinib can be taken with or without food. Because acalabrutinib may be metabolized by CYP3A4, participants should be strongly cautioned against consumption of grapefruit, grapefruit juice, or Seville orange juice (which contain potent CYP3A4 inhibitors) or using herbal remedies or dietary supplements (in particular, St John’s Wort, which is a potent CYP3A4 inducer). Otherwise, participants should maintain a normal diet unless modifications are required to manage an AE such as diarrhoea, nausea or vomiting.\n\nContraception\n\nContraception is mandated from trial entry until 12 months after the last treatment dose for participants of reproductive potential.\n\nParticipants with reproductive potential must use two highly effective methods of contraception during the study, with male participants also agreeing to use additional barrier or abstinence due to the risk of exposure to a developing foetus if the female partner is already pregnant.\n\nHormonal contraception may be susceptible to interaction with study or other drugs, which may reduce the efficacy of the contraception method.\n\nBoth male and female patients must be counselled about future fertility prospects and sperm/ovarian preservation prior to study entry. Male patients must refrain from donating sperm once on study and during treatment and the 12 month follow-up period. Acalabrutinib is not known to confer long term reduction in fertility, however the cytotoxic drugs used in this protocol, i.e. doxorubicin and cyclophosphamide, may do.\n\nProvisions for post-trial care\n\nParticipants completing the trial, or who withdraw their consent for follow-up, will be managed by standard clinical care from their usual treating clinician.\n\nPrimary outcomes\n\nThe Phase I study will be dose escalation that will proceed until an MTD is defined or the MAD is determined in order to define the RP2D of acalabrutinib in combined with R-CHOP. This dose will be used in the Phase II evaluation of acalabrutinib in combination with R-CHOP in participants with DLBCL.\n\nThe DLT will be defined using the National Cancer Institute (NCI) Common Terminology Criteria for Adverse events (CTCAE) version 4.03. The MTD is defined as the highest dose level at which none of the first six treated participants, or no more than 2 of the first 12 treated participants, experiences a DLT.\n\nThe DLT reporting period is from the start of treatment until the end of cycle 3. Events meeting the below criteria following the end of cycle 3 will not be recorded as DLTs.\n\nA DLT will be defined as the occurrence of the following:\n\n- Grade 2 or greater haemorrhagic event requiring medical intervention or any intracranial haemorrhage\n\n- Grade 3 or greater non-haematological toxicity at least possibly related to acalabrutinib (including grade 3 or 4 biochemical AEs). The following will be excluded; Grade 3 or 4 nausea in participants who have not received optimal treatment with anti-emetics, Grade 3 or 4 diarrhoea in participants who have not received optimal treatment with anti-diarrhoeal therapy, alopecia and those participants experiencing grade 3 or 4 administration reactions from rituximab.\n\n- Grade 4 thrombocytopenia or neutropenia for more than 7 days despite GCSF use.\n\n- Any complete, continuous dose interruption more than 7 days for acalabrutinib related toxicities of grade 2 or greater within cycle 2.\n\nIf a DLT attributed to acalabrutinib per the investigator’s assessment occurred, dosing with acalabrutinib will be withheld. Acalabrutinib treatment will be resumed at a lower dose only after toxicity has resolved to ≤ grade 1. There will be no dose re-escalation for acalabrutinib after recovery from toxicity, and no intra-cohort participants dose escalation will be allowed. The study will terminate early if three DLT occur for the first six participants in Phase I (Table 4).\n\nDose adjustments for R-CHOP components, will follow conventional dose modification schedules.\n\nPhase II will use ORR to document the anti-tumour activity of the acalabrutinib and R-CHOP combination therapy, as well as investigating treatment safety. Safety will be assessed throughout via:\n\nThe reporting of adverse events using the NCI CTCAE v4.03.\n\nRegular haematological and biochemistry evaluation\n\nDLT defined using the NCI CTCAE v4.03.\n\nSecondary outcomes\n\nPharmacokinetics of acalabrutinib, area under the curve (AUC), maximum concentration (Cmax), time to maximum concentration (Tmax), half-life (T1/2) and other PK parameters\n\nOverall response rate of the combination acalabrutinib and R-CHOP according to cell of origin\n\nProgression-free survival at 2 years\n\nOverall survival (death from any cause) at 2 years\n\nTertiary outcomes\n\nBtk occupancy by acalabrutinib on peripheral blood using fluorescent affinity probe assay\n\nAntibody-dependent cell-mediated cytotoxicity of R-CHOP when combined to acalabrutinib, post 1st R-CHOP and at day 8, 2nd cycle acalabrutinib + R-CHOP\n\nCD86 and CD69 expression as a function of BCR activation by flow cytometry.\n\nTumour-specific DNA in plasma will be sequenced throughout treatment and clinical course\n\nApply the following techniques to formalin fixed paraffin embedded (FFPE) tumour material: mutational panel, Fluorescence In Situ Hybridization (FISH) analysis, immunohistochemical analysis for dual protein expression of Myc and Bcl2 and gene expression profiling using whole transcriptome profiling\n\nThe time schedule of enrolment, interventions, assessments, and visits for participants are fully detailed in the schedule of events (Table 2).\n\nThere is no formal sample size calculation given the modified 6+6 classical design. Sample size for Phase I is based upon anticipated numbers to complete schedule of dose escalation to DLT or MAD (n=6 - 24 participants). For Phase II, the sample size (n=15) permits sufficient numbers of participants to gain additional safety information and to look for exploratory signal of efficacy in biological subgroups in a pooled analysis of both stages.\n\nPatients are approached within a hospital setting and screened for eligibility by research staff to ensure all inclusion and exclusion criteria are met. Clinicians will seek informed consent to enter the trial from a patient only after the patient has received a full explanation of the trial, the patient has read the PIS and had enough time to consider taking part.\n\nAfter the participant has entered the trial the clinician remains free to give alternative treatment to that specified in the protocol at any stage if he/she feels it is in the participant’s best interest, but the reasons for doing so should be recorded. In these cases, participants will be withdrawn from protocol treatment but remain within the trial for the purposes of follow-up and data analysis.\n\nACCEPT is a non-randomised open-label study, consequently there is no allocation sequence and no-one involved in the trial is blinded to the intervention.\n\nPlans for assessment and collection of outcomes\n\nHospital research staff will enter participant data into the study eCRFs via a remote data collection tool (Medidata Rave). Only trained personnel with specific roles in the study will be granted access to the eCRFs. SCTU trial staff will regularly check the data for missing or anomalous values. Data queries will either be automatically generated within the eCRF, or manually raised with site by the SCTU team. Site staff will respond to explain or resolve the discrepancies.\n\nPlans to promote participant retention and complete follow-up\n\nACCEPT has no specific plans for promoting retention to the trial. Participants who do not complete six cycles of treatment for reasons other than toxicity will be replaced.\n\nData management\n\nFull details of the data management strategy for the study are available as Extended data14.\n\nConfidentiality\n\nParticipant data is pseudonymised by assigning each participant a participant identifier code, which is used to identify the participant during the study and for any participant- specific communication between SCTU and site.\n\nPharmacokinetic sampling\n\nAcalabrutinib pharmacokinetics will be assessed in all participants by determination of serum concentrations of acalabrutinib by a fully validated assay that has already been used in human studies. The samples will be taken from participants as explained in Table 5.\n\naPost dose samples should be taken at the above specified times ± 5 minutes after study drug administration\n\nPharmocodynamic sampling\n\nAcalabrutinib pharmacodynamics will be assessed in all participants by measurements of the following:\n\n1. Evaluation of BTK occupancy in PBMCs.\n\nPBMC will be incubated with a fluorescently-tagged analogue of acalabrutinib. Acalabrutinib and the probe bind covalently to Btk, with binding of the probe being prevented by acalabrutinib occupancy. Probe-binding will be detected by flow cytometry. PMBCs will be collected by blood sample on day 1 of cycle 2 pre-dose and at 4 hours post-dose and on day 8 of cycle 2 pre-dose, at 2 and 4 hours post dose (Table 6).\n\nADCC, Antibody dependent cellular cytotoxicity; BTK, Bruton Tyrosine Kinase; PBMC, Peripheral Blood Mononuclear cells\n\naThe post dose sample should be taken 4 hours ± 5 minutes after study drug administration.\n\n2. Impact of the addition of acalabrutinib on R-CHOP mediated ADCC\n\nBlood samples will be collected on day 1 and day 8 cycle 2 pre- dose and 4 hours post dose. A fluorescent dye release assay measuring target cell lysis, coupled with flow cytometry will be employed to measure ADCC (Table 6).\n\n3. BCR activation before and after R-CHOP + acalabrutinib\n\nBlood samples for measurement of BCR activation will be collected on day 1 and day 8 cycle 2 pre-dose and on day 1 cycle 4 pre-dose. BCR activation will be determined by measurement of CD86 and CD69 expression on PBMC by flow cytometry (Table 6).\n\nTranslational research\n\nThis study will prospectively validate the cell of origin (ABC versus GCB) model of DLBCL and its practicality and utility, as well as assessing the benefit/toxicity of the addition of acalabrutinib to R-CHOP.\n\nFor all participants, sufficient diagnostic material should be available to forward to a central laboratory for pathological review and gene expression profiling. FFPE lymph node biopsies should be forwarded to Haematological Malignancy Diagnostic Service (HMDS) where molecular phenotyping will be performed using a reliable array platform requiring the extraction of RNA. Central pathological review will be performed as quality check but not fed back to site unless findings untoward. There must be sufficient tumour block to test for H/E morphological check of compatibility of DLBCL diagnosis, immunophenotyping, RNA for gene expression profiling. Additional material will be used for immunohistochemical, cytogenetic and molecular genetic studies FISH, DNA for mutation, Transcription Mediated Amplification. Material will only be retained if the sample size is sufficient for removal of material without compromising its value as archived diagnostic material. This may include the generation of extended full section immunohistochemical studies, tissue microarrays and DNA extraction. The remaining FFPE block will be returned to the local pathologist.\n\nDNA extracted from tumour material will be used to perform mutation detection on BCR pathway (e.g. Btk, PI3K, CD79b). Genetic abnormalities of BCR pathway will be correlated with clinical outcomes and the expression of BCR pathway target genes.\n\nStatistical methods for primary and secondary outcomes\n\nA full and detailed statistical analysis plan will be developed prior to the final analysis of the trial. The main features of the statistical analysis plan are:\n\nParticipants are required to receive 6 cycles of treatment to be included in the analysis, unless the cycles are not completed due to toxicity. The safety population will include all participants who receive any component of therapy.\n\nBaseline characteristics will be summarised with means and standard deviations for continuous outcomes (if data is skewed medians and ranges will be presented) and frequencies and percentages for binary outcomes.\n\nThe primary analysis will be presented in a descriptive fashion, including a summary of any DLTs experienced by participants in Phase I and summary statistics for overall response rates for participants in Phase II, summarised with frequencies and percentages.\n\nThe safety component of the primary analysis will consist of all AEs and SAEs summarised by the CTCAE System Organ Class and term. SAEs by relatedness will also be listed.\n\nKaplan-Meier survival curves will be produced for the time to event secondary outcomes of progression-free survival and overall survival.\n\nThe final analysis will be conducted after one of the following conditions is met.\n\n○ The trial is terminated early (for example, due to toxicity).\n\n○ All participants have had the opportunity to complete protocol defined therapy and have completed the final follow-up visit, which will be 2 years after receiving the last study treatment, or sooner, if all participants have progressed, died or withdrawn from the study.\n\n○ The predicted date for the final analysis is quarter 3 of 2022\n\nInterim analyses\n\nIn Phase I the Safety Review Committee will review safety data and advise on dose escalation. The study will terminate early if three DLT occur for the first 6 participants in Phase I.\n\nIn Phase II an independent Data Monitoring & Ethics Committee (DMEC) will review outcome and safety data regularly during the trial advising the independent Trial Steering Committee (TSC) on continuation of the trial.\n\nMethods for additional analyses (e.g. subgroup analyses)\n\nNot applicable.\n\nMethods in analysis to handle protocol non-adherence and any statistical methods to handle missing data\n\nIf the participant is discovered to be ineligible over the course of the study their participation will end. Where possible, participants who have withdrawn from trial treatment should remain in follow-up as per the trial schedule. If participants additionally withdraw consent for this, they should revert to standard clinical care as deemed by the responsible clinician. It would remain useful for the trial team to continue to collect survival follow-up data and unless the participant explicitly states otherwise, survival follow-up data will continue to be collected. Details of trial discontinuation (date, reason if known) should be recorded in the eCRF and medical record.\n\nPlans to give access to the full protocol, participant level-data and statistical code\n\nThe full protocol is available as Extended data14. The ‘Dissemination plans’ section of this article describes how to access trial-related data.\n\nComposition of the coordinating centre and trial steering committee\n\nAn independent TSC has been set up to monitor trial progress. The ACCEPT TSC charter defines the membership, terms of reference, roles, responsibilities, authority, decision-making and relationships of the TSC, including the timing of meetings, frequency and format of meetings and relationships with other trial committees.\n\nThe ACCEPT Trial Management Group includes representatives with expertise in haematology, oncology, translational science and medical statistics; as well as being supported by a Patient and Public Involvement contributor and CTU staff involved in the day-to-day running of the trial. Charters for these groups are available via accept@soton.ac.uk.\n\nComposition of the data monitoring committee, its role and reporting structure\n\nAn independent DMEC comprising three clinicians and a statistician experienced in this research area (but not directly involved in this trial apart from DMEC membership) has been set up to monitor trial progress and safety. The Charter for this group is also available via accept@soton.ac.uk.\n\nAdverse event reporting and harms\n\nData on AEs will be collected at treatment and follow-up visits. SAEs will be reported up to 30 days after the last administration of the trial drug to the SCTU safety desk. The trial also has a UK regulatory compliant real-time SAE reporting process to identify serious AEs and suspected unexpected SAEs that could suspend or stop the trial if warranted.\n\nFrequency and plans for auditing trial conduct\n\nThe SCTU has undertaken a risk assessment for the ACCEPT trial, which includes the requirements for monitoring (both central and site). The SCTU undertakes a number of internal audits of its own systems and processes annually and has routine audits from both its sponsor and the independent Medicine and Health care products regulatory authority (MHRA).\n\nPlans for communicating important protocol amendments to relevant parties (e.g. trial participants, ethical committees)\n\nResearch ethics committee/MHRA-approved protocol amendments will be communicated to sites via email and updated trial documentation provided centrally via the SCTU trial website (https://www.southampton.ac.uk/ctu). Trial registries will be amended where relevant with explanations for these changes.\n\nThe end of the study is defined as the date of the last follow-up visit of the last participant (expected to occur 24 months after the last participant receives their last study treatment). NB: The study will terminate early if three DLT occur for the first 6 participant in Phase I.\n\n\nDissemination plans\n\nThe results from the ACCEPT trial will be disseminated to patients and clinical teams through peer-reviewed journal articles authored by the members of the trial management group and presented at international conferences. We will also publicise our findings through existing networks and patient groups. Summary trial results will be available on the SCTU website.\n\nPseudonymised individual participant data within the clinical trial dataset will be available for sharing via controlled access by authorised SCTU staff (as delegated to SCTU by the trial sponsor). Data access can be requested via a SCTU Data Release application form; detailing the specific requirements and the proposed research, statistical analysis, publication plan and evidence of research group qualifications. Please email the completed form to the SCTU Data Release Committee Coordinator at ctu@soton.ac.uk.\n\nData access requests are reviewed against specific eligibility criteria by the SCTU data custodian and key members of the trial team, including a statistician and chief investigator or by an external Independent Review Panel. Decisions about requests are made promptly and usually no more than three months after receipt of request. Responses to all data requests, with a clear rationale for any refusals, will be sent promptly to the data requester.\n\n\nTrial status\n\nRecruitment opened in May 2017 and finished January 2020; the trial is now closed to recruitment and is in follow-up. However, if any of the current participants (n=9) withdraw prior to receiving 6 cycles of R-CHOP, for reasons other than toxicity, we will need to replace them. If this situation arises, we will re-open recruitment to all sites.\n\nIf the trial does not need to reopen, the last participants will reach cycle 6 on 20 April 2020 and will complete treatment on 6 July 2020. Participants enter a 24-month follow-up period after treatment and LPLV will be July 2022. The Phase I participants are nearing the end of their follow-up period and FPLV is due on 15 Feb 2020.\n\nThis clinical trial was entered into EudraCT in December 2017 (EudraCT Number: 2015-003213-18) and is registered as ISRCTN 13626902.\n\n\nDiscussion\n\nACCEPT aims to find out the maximum dose of acalabrutinib that can safely be given to patients in combination with R-CHOP as well as investigating the potential side effects and activity in patients with DLBCL.\n\nThis is the first use of the second generation Btk inhibitor acalabrutinib in combination with R-CHOP. This has been addressed through use of an appropriate Phase I trial design and monitoring during the study. The protocol contains stopping criteria in response to side effects which may occur with dose levels. To maintain safety during the trial, the data will be monitored closely by a Safety Review Committee and an independent DMEC on an on-going basis. First generation Btk inhibitors have been used safely in combination with R-CHOP in this patient group.\n\nPrevious preclinical and clinical observations make a compelling case for the clinical investigation of R-CHOP in combination with acalabrutinib for the treatment of patients with DLBCL. However, the clinical effects of acalabrutinib on DLCBL are unknown and the initial group of patients in particular are unlikely to derive benefit from low dose acalabrutinib. It will be made clear to all patients that the benefit of acalabrutinib is uncertain.\n\nMultiple blood samples will be taken in this study. Participants may also be subjected to up to two bone marrow biopsies and a lumbar puncture. These study procedures have been designed to be as limited as possible to gain the necessary information and to be conducted in the least distressing way by experienced health care professionals.\n\nParticipants will have two PET/CT scans and a separate CT with contrast (Cycle 7). These are standard of care in DLBCL. In addition, they will undergo two additional CT scans to look for progression as a research investigation (months 12 and 24). Patients will be made fully aware of this and the risks of these scans prior to giving consent to their participation in the trial.\n\nIn addition, an Echocardiogram or MUGA will be performed for all patients to establish whether patients have a left ventricular ejection fraction of >55% to ensure they are suitable to receive 300mg/m2 doxorubicin.\n\nThere are no particular legal concerns arising from this study. The SCTU at the University of Southampton will be the UK coordinating trials unit and the University Hospital Southampton NHS Foundation Trust the sponsor in the UK and responsible for all legal requirements of conducting a non-commercial clinical trial of an investigation product. The drug for this trial is provided by ACERTA Pharma, BV who are responsible for the Good Manufacturing Practice quality drug and who is also providing research funding.\n\n\nData availability\n\nNo underlying data is associated with this article.\n\nFigshare: ACCEPT - combining acalabrutinib with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP) for Diffuse Large B-cell Lymphoma (DLBCL): study protocol for a phase Ib/II open-label non-randomised clinical trial, https://doi.org/10.6084/m9.figshare.11791275.v414.\n\nThis project contains the following extended data:\n\n- Informed Consent Form and Patient Information Sheet – Tissue Block Screening; Phase I; Phase II; Pregnancy\n\n- Full trial protocol\n\n- Data Management Plan\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFigshare: SPIRIT checklist for ‘ACCEPT - combining acalabrutinib with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP) for Diffuse Large B-cell Lymphoma (DLBCL): study protocol for a phase Ib/II open-label non-randomised clinical trial’, https://doi.org/10.6084/m9.figshare.11791275.v414.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nEthics approval and consent to participate\n\nThe study received ethical approval from South Central - Berkshire Research Ethics Committee on 26 January 2017 (ref: 16/SC/0657) and has Health Research Authority approval (IRAS 182046). Written informed consent for participation and publication of the participants/patients’ data was obtained from the participants. Participants are free to withdraw at any time. The Informed Consent Forms are provided as Extended data.\n\n\nConsent for publication\n\nResponsibility for publication has been delegated to Andrew Davies (Chief Investigator) and Gareth Griffiths (Director of SCTU) who have consented to this publication.\n\n\nSponsor information\n\nName and contact information for the trial sponsor: University Hospital Southampton NHS Foundation Trust (email: R&Doffice@suht.swest.nhs.uk), sponsor reference number: RHM CAN 1129.\n\nThe study sponsor was not involved in the study design; writing of the protocol paper; or the decision to submit the paper for publication.", "appendix": "References\n\nCancer Research UK: UK Non-Hodgkin’s Lymphoma Statistics. (Accessed February 2019). Reference Source\n\nA clinical evaluation of the International Lymphoma Study Group classification of non-Hodgkin’s lymphoma. The Non-Hodgkin’s Lymphoma Classification Project. Blood. 1997; 89(11): 3909–18. PubMed Abstract \| Publisher Full Text\n\nFisher RI, Gaynor ER, Dahlberg S, et al.: Comparison of a standard regimen (CHOP) with three intensive chemotherapy regimens for advanced non-Hodgkin’s lymphoma. N Engl J Med. 1993; 328(14): 1002–1006. PubMed Abstract \| Publisher Full Text\n\nCoiffier B, Lepage E, Bière J, et al.: CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N Engl J Med. 2002; 346(4): 235–242. PubMed Abstract \| Publisher Full Text\n\nHabermann TM, Weller EA, Morrison VA, et al.: Rituximab-CHOP versus CHOP alone or with maintenance rituximab in older patients with diffuse large B-cell lymphoma. J Clin Oncol. 2006; 24(19): 312–7. PubMed Abstract \| Publisher Full Text\n\nPfreundschuh M, Schubert J, Ziepert M, et al.: Six versus eight cycles of bi-weekly CHOP-14 with or without rituximab in elderly patients with aggressive CD20+ B-cell lymphomas: a randomised controlled trial (RICOVER-60). Lancet Oncol. 2008; 9(2): 105–116. PubMed Abstract \| Publisher Full Text\n\nPfreundschuh M, Trumper L, Osterborg A, et al.: CHOP-like chemotherapy plus rituximab versus CHOP-like chemotherapy alone in young patients with good-prognosis diffuse large-B-cell lymphoma: a randomised controlled trial by the MabThera International Trial (MInT) Group. Lancet Oncol. 2006; 7(5): 379–391. PubMed Abstract \| Publisher Full Text\n\nSehn LH, Donaldson J, Chhanabhai M, et al.: Introduction of combined CHOP plus rituximab therapy dramatically improved outcome of diffuse large B-cell lymphoma in British Columbia. J Clin Oncol. 2005; 23(22): 5027–5033. PubMed Abstract \| Publisher Full Text\n\nGisselbrecht C, Glass B, Mounier N, et al.: R-ICE versus R-DHAP in relapsed patients with CD20 diffuse large B-cell lymphoma (DLBCL) followed by autologous stem cell transplantation: CORAL study. J Clin Oncol. 2005; (27): abstr. 8509. Reference Source\n\nDavis RE, Ngo VN, Lenz G, et al.: Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma. Nature. 2010; 463(7277): 88–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYounes A, Thieblement C, Morschahauser F, et al.: Combination of ibrutinib with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) for treatment-naive patients with CD20-positive B-cell non-Hodgkin lymphoma: a non-randomised, phase 1b study. Lancet Oncol. 2014; 15(9): 1019–26. PubMed Abstract \| Publisher Full Text\n\nBarf T, Kaptein A: Irreversible protein kinase inhibitors: balancing the benefits and risks. J Med Chem. 2012; 55(14): 6243–6262. PubMed Abstract \| Publisher Full Text\n\nLandau DB, Hughes L, Baker A, et al.: IDEAL-CRT: A Phase 1/2 Trial of Isotoxic Dose-Escalated Radiation Therapy and Concurrent Chemotherapy in Patients With Stage II/III Non-Small Cell Lung Cancer. Int J Radiat Oncol Biol Phys. 2016; 95(5): 1367–77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDavies A, Ardeshna K, Barrans S, et al.: ACCEPT - combining acalabrutinib with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP) for Diffuse Large B-cell Lymphoma (DLBCL): study protocol for a phase Ib/II open-label non-randomised clinical trial. figshare. Journal contribution. 2020. http://www.doi.org/10.6084/m9.figshare.11791275.v4\n\nhttps://clinicaltrials.gov/ct2/show/NCT02029443" }	[ { "id": "69037", "date": "01 Oct 2020", "name": "Allan Hackshaw", "expertise": [ "Reviewer Expertise Cancer 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\nA well-designed and efficient phase I then II trial. The phase I stage involves finding whether once or twice daily acalabrutinib has acceptable safety. This then rolls into a single arm phase II trial. All of the endpoints are in common use. This project also has a key translational component, making this trial more informative at the end.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] }, { "id": "72306", "date": "12 Oct 2020", "name": "Wendy Osborne", "expertise": [ "Reviewer Expertise Clinical and biological lymphoma" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nRationale clear and an important unmet need to address, in view of the poor outcomes for patients with R/R DLBCL. Biological evidence already available to confirm dysregulation of BCR signalling and in agreement with rationale for a second generation Btk inhibitor.\n\nTable 1 is clear and of value to the manuscript.\n\nFigure 1 is clear and well designed, end of treatment CT is listed in this figure but standard of care 2 PET scans and a separate CT is described in ionising radiation paragraph. Imaging to be clarified on figure 1.\n\nInclusion and exclusion criteria as standard for target population.\n\nDose limiting toxicities are also well defined and appropriate for this combination and design.\n\nTranslational research is clearly described and of value to the aims of the study, pathology review is performed and essential to have within this trial design. There is clear and appropriate governance structure for the trial.\n\nIn summary, excellent rationale and design with no concerns about protocol, imaging use to be clarified on figure 1.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-941
https://f1000research.com/articles/9-660/v1	30 Jun 20	{ "type": "Research Article", "title": "Measures to increase value of preclinical research - an inexpensive and easy-to-implement approach to a QMS for an academic research lab", "authors": [ "Michael Hewera", "Ann-Christin Nickel", "Nina Knipprath", "Sajjad Muhammad", "Xiaolong Fan", "Hans-Jakob Steiger", "Daniel Hänggi", "Ulf Dietrich Kahlert", "Ann-Christin Nickel", "Nina Knipprath", "Sajjad Muhammad", "Xiaolong Fan", "Hans-Jakob Steiger", "Daniel Hänggi" ], "abstract": "Background: Increasing concerns emerge regarding the limited success in reproducing research results. This is a major problem for science, society and economy. Driven by industry or scientific networks, several attempts to combat this crisis are initiated. However, only few measures address the applicability and feasibility of implementation of actions into an academic research environment with limited resources. Methods: Here we propose a strategy catalogue aiming for a quality management system suitable for many research labs. Our proposal is guided by its inexpensiveness and possibility of rapid installation. Moreover, we restrict presentations of our actions on those for what we received a positive feedback by the users regarding its applicability. For this we used eLabFTW, an electronic lab book, as hub for all other components of our quality management system (QMS). Storage of lab journals and project management will be done there as well. Standard operation procedures have been introduced. Those will be stored in eLabFTW too. Furthermore, we implemented a bio bank for safer long term storage of bio samples and cryo-cultures of cell lines. Next we set up a lab meeting as feedback mechanism for the QMS. In a final step we implemented an automated pipeline to be used for example for drug screens. Results: With this effort we want to reduce individual differences in work techniques, to further improve the quality of our results. Although, just recently established, we can already observe positive outcomes in quality of experimental results, improvements in sample and data storage, stakeholder engagement and even promotion of new scientific discoveries. Conclusions: We believe that our experiences can help to establish a road map to increase value and output of preclinical research in academic labs with limited budget and personnel.", "keywords": [ "QMS", "Quality Management", "SOP", "Automation", "Lab book", "electronic lab book", "Cell banking", "database" ], "content": "Background and Goal\n\nDue to its immense documentation load, the establishment of a quality management system (QMS) certified with ISO 9001 standard1 in biomedical research labs is challenging. This is particularly true for academic labs where usually staff is changing rapidly and the progress for individual careers is often the dominator for operator’s decision making. However, it is shown, that a lack of quality management and transparency are two reasons for the ongoing reproducibility crisis2–4. We implemented a slim line QMS that should lead to an increase of confidence in our scientific lab outcomes by optimizing internal processes for elevated transparency and reproducibility.\n\n\nMethods\n\nWe introduced several technological tools to optimize processes, which combined, will form the QMS (Figure 1) of our lab.\n\nSOP: Standard operation procedure, QMS: Quality management system.\n\n\nExperience with freeware solution for electronic lab documentation\n\nTransparent data management is a cornerstone for research integrity. As the basis of our work we set up an electronic documentation system to document all lab activities of all lab members. We chose eLabFTW5 in the current version 3.4.8, an open source solution to track experiments with a powerful and flexible database. In the used version it provides all features necessary to provide the grounds of conformity with the principles of good research practices as proposed by the German research Foundation (DFG). This includes, not exclusively, the installation of a non-delete policy for created entries. The relevant software code can be found on GitHub platform6. eLabFTW is made up from two parts. One is called ‘experiments’ and is the lab journal part of the software, the other is called ‘database’ and lets the user create text entries in self defined data types. The journal part of eLabFTW will be used by each researcher to document their research, and for project management. Created entries cannot be deleted and changes to these entries will automatically be logged. The database part will be the backbone of our QMS. We decided to share reading and search authorization for all entries amongst all registered personal accounts in our working group to increase transparency and to stimulate the critical interaction. Data storage, backup and archiving of eLabFTW data files can be easily implemented in regular executed local data management policy. All finished experiments will be time stamped (digital signature), and will be blocked for editing from any user by the server itself. This will secure the written text and attached primary data (pictures, raw data from lab machines etc.) from later manipulations, as they are now unchangeable and undeletable.\n\n\nEstablishment of coherent protocol and lab tool documentation system\n\nSOPs (standard operating procedures) are a main principle of most QMS. To our experience, even within one working unit, for one given experiment various self-developed, self-adapted protocols are in use at the same time. By implementing a binding SOP policy for all users, defining the accountability of the data to qualify for the use in external presentation such as in a scientific publication, we aim to enhance process coherence in order to increase intra-lab reproducibility success. As first step, a template for SOPs was created and its categorization and storage were implemented in the electronic lab documentation system followed by the establishment of a writing procedure for how to complete the template was developed. Any person can then write a draft of a new SOPs, but creation of the SOPs and QMS implementation are managed by selected authorized identity (in our hands a governmental- certified technical assistant to ensure conformation with regulation processes), to avoid duplicates or development of multiple parallel versions of one subjects. Approved, newly established SOPs then are categorized by name, a standardized alphanumerical identifier, and version number. Then all new SOPs get uploaded to the database of the electronic lab book. If applicable, SOPs are electronically linked with each other if their execution/specification depends on each other. In the last year 91 unique procedures and recipes have been standardized. 31 of them have been revised at least once, to either correct them, or adapt them to new findings or necessities. Every new employee will be sent a package of our most used methods and receipts as well as SOPs related to his proposed project, prior to their first day of work, so they have the chance to already get familiar with the methods in theory.\n\nMoreover, virtual one-to-one project development meetings have been integrated in our e-documentation system by weekly, time-stamped progress reports of each lab member. Those are composed of structure items common for academic research projects featuring presentation of new results, problems experienced, suggestions of problem management and update on current literature. Leadership provides feedback on each parameter within a timely manner to secure up-to-date project management. Importantly, apart our open visibility of progress reports and feedback to all team members ensuring full transparency for all stakeholders, we have set up visibility-restricted sub projects. Those more restricted groups are intended to exchange work-in–progress documents between the individual user and the leadership, that have certain impact on the “outside-presentation” of the lab, such as presentations or manuscript drafts. We found this virtual project development-meeting platform enables efficient time management of the leadership and has been proven suitable to be conform with guidelines to minimize social contact, that have been stated by governments worldwide to fight the Covid-19 pandemic.\n\n\nBio Bank\n\nAccording to FAIR principles, when publishing research results, used material and created bio samples (tissue, cell pellets, DNA, RNA, plasmids, etc.) need to be held in stock to allow for future replications of the experiments performed or to share lab tools with the community. By establishing our lab bio bank, we aimed to provide a centralized storage facility for bio samples both for experimental and clinical research projects. The bio bank consists of a -80°C deep freezer and a liquid nitrogen tank. Both contain labelled boxes with designated positions for bio samples. Stored samples are characterized by a unique identifier. Long term preservation is supported by using freezing approved label technology and designation with printing instead of handwriting. The entire needed lab infrastructure to install our bio bank is a standard set up in each lab and does not require extra investments. Due to the anticipated large and constantly increasing number of bio samples included in the storage, we decided not to integrate the storage data into the database of eLabFTW but used a separate SQL database as the digital complement of the physical bio bank. Nevertheless, samples and experiments can be cross referenced in both platforms using the unique identifiers of the samples in the experiment section of eLabFTW, thereby securing complete and intertwined electronic documentation in our QMS. All lab members can search for samples in the digital database. However only three people have write access and can add and delete entries. Only those three people can access the physical bio bank, to add and remove samples. With this setup we aim to minimize sample loss and mix up, as well as ensure proper labelling of the samples.\n\n\nQuality assessment of cell models\n\nAs for many research labs, most of our projects fundament on in vitro experiments. For most of the cancer research projects, we perform our work by using the standard disease model type: the cancer cell line. Meanwhile paying particular attention to create the most-accurate-as-possible recapitulation of the pathophysiology of the disease by using 3D cultures, we furthermore surveil our technical quality of the applied biological test matrixes. This includes a) the confirmation of authentication through short tandem repeat analysis (STR) of cultured models every 6 months, b) the use of as-young-as-possible cell models (monitoring cell passage number); c) surveillance and - if necessary - clearance of mycoplasma contamination (PCR-based mycoplasma detection) and d) adherence to culture procedures as defined in SOPs. The STR analysis is outsourced to an in-house service. There Quantitation was performed using the QuantusTM Fluorometer (Promega) and the QuantiFluor® dsDNA Sample Kit (Promega) following manufacturer’s instructions. A multiplex PCR of 21 STR loci (PowerPlex® 21 System Kit, Promega) was performed in a total reaction volume of 12.5 µl with 0.5 ng template DNA. Thermal cycling conditions were followed as described by the manufacturer. Capillary electrophoretic separation was performed on the ABI Prism® Genetic Analyzer 3130 equipped with a 36 cm Capillary Array/POP-4 (Applied Biosystems, Darmstadt, Germany) following manufacturer’s instructions. Data acquisition and analysis was performed using the ABI Prism 3130 Collection software (Applied Biosystems) and GeneMapperID® v.3.2 software (Applied Biosystems, Darmstadt, Germany). For STR and mycoplasma test we invest less than 20 USD in total. We believe that the adherence to chronic execution of those simple methods is an economical, feasible and robust measure to sustainably reduce the creation of low-value cancer cell line data.\n\n\nFeedback and update communication platform\n\nA monthly gathering of all participating and interested stakeholders secures the communication of updates and feedback mechanism of the QMS. The date and location for the meeting is circulated well in advance to ensure high frequency of participation of invitees. The feedback platform is open for all stakeholders independently of profession, profession hierarchy and amount of involvement in hands-on lab work. Each time a protocol of the meeting is generated in form of meeting minutes-like listing including specification of attendance. Although sometimes time consuming due to extent exchange of opposing opinions on topics, we hypothesize the impact of this outreach effort to involve all participants in the policy development extends further then the QMS. We believe such a platform increases the teamwork atmosphere and group belonging hereby increasing work atmosphere. We also think this is a valuable tool to fight occurrence of inequality and discrimination in our working group. The generated meeting minutes will be used to permanently alter the QMS to guarantee the highest possible quality. For that they will also be uploaded to the electronic lab book.\n\n\nAutomation for lab work execution\n\nAutomation is a standard in industry labs and clinical diagnostics. We implemented a robotic liquid handling instrument (Beckman Coulter Biomek FxP) connected with automated plate reading for performing pharmacology testing on a cancer cell (Vargas-Toscano et al.)7. We chose this method as our implementation trial since this is one of our most frequently used lab procedures. Our validation experiments based on manual repetition of the experiment proves that the functionality of our automation assay. Besides the accurate execution, the possibility of direct data reporting into the electronic lab documentation system and the possibility of electronic surveillance of executed pipetting for error reduction makes the inclusion of automation a valuable measure to increase reproducibly and transparency of our work. We hypothesize that robotic assays increase the value and innovation of our work. As such, the application of the screening identified a repurpose of a FDA approved neurotransmitter drug to inhibit growth of brain tumor cells. Given the recent high-profile publications revealing the importance of neurotransmitter signaling for the biology of brain cancer8, our QMS coincidently enabled us to contribute to current line of cancer research that we had not purposely addressed without it. Given the existence of low-cost pipetting automation, more economically feasible options instead of the Biomek solution are available. Here, single-dose pumps in particular come to mind. These are of cause not as comfortable as robotic systems but fulfill the need of standardized pipetting. Used pumps can be bought for less than 1000 USD.\n\n\nResults and discussion\n\nIn our experience the approach of using an all-digital lab book with shared reading rights and automated storage of raw data, does not only guarantee data management according to FAIR9 or increase the transparency according to the Hong Kong principles10, but also helps to increase motivation of the team in general and thereby improving scientific development of the users. From all 17 lab members (at the time of publishing this), only 4 are not using the electronic lab book for recording their experiments. Those are members who are at the end of their projects and were therefore encouraged to keep their old method of recording, for consistency. Yet also those people use the database part of the electronic lab book, as well as the SOPs.\n\nThe use of SOPs, which were uploaded to the electronic lab book, lead to the standardization of particular processes in the lab. Before researchers performed several experiments with protocols from other or former labs. This leads to lack of reproducibility inside of the lab. Now a higher consistency of experimental results is given, as all researchers perform each experiment in only a single way, minimizing individual differences.\n\nCompared to shared freezers used in the lab, central administration by only a few people highly reduces the loss of samples and mix-ups. Furthermore, the bio bank and its digital complement, with the possibility of linking it to the electronic lab book, make it very easy to find all samples used in all experiments. Since the Bio Bank is relatively freshly implemented, we cannot give results for the improvement of storage over longer periods of time yet. Until now the model of a digital copy of the bio bank, combined with limited access to the physical bank is holding up. All samples that got stored in the bio bank are still unmistakably findable. We argue that due to the QMS we fulfill the requirements for storage of data and samples, according to good scientific practice guidelines, as proposed by DFG11.\n\nSTR and regular mycoplasma testing lead to rejection of low quality cell cultures, ensuring higher significance of results derived from experiments with those cells (Table 112). Especially for metabolics or pharmacoproteomics experiments, a contamination with mycoplasma can lead to wrongful results. In our experience, even the most skillful and experienced cell culture scientists cannot avoid the introduction of unrecognized contamination. With the introduced QMS, in our lab we identified cancer cell models have established subclones with different genetic background due to inter cell line contamination. Those subclones were designed as a different cell line instead of a subclone. The introduction of the QMS leads to significant project adjustments and in some case to termination of experiments. We also put on hold a scheduled submission of a manuscript draft for publication due to cell line miss-identification.\n\nFor comparison and a reference for the scientific community, STR profiles of parental lines are provided as well. STRs with mixes of both cell lines are marked. For original data as supplied by in-house service, see underlying data12.\n\nThe implemented communication system already proved its worth by leading to updated and refined versions of some SOPs. Given the deliverables of the meetings have significant influence on future lab procedures, we have experienced a significant increase in involvement and commitment of responsible lab members in this effort. A regular non-science oriented lab meeting creates an opportunity for easier expression of general matters thereby facilitating interpersonal communication amongst stakeholders. The virtual one-to-one project development report-feedback loops provides an opportunity to timely manage diverse research branches with moderate resources needed at each time.\n\nAs the technologically most advanced component of our QMS, we implemented an automated pipetting system in our lab routines. We believe that the robustness and transparency of the data generation, as well as the simplicity of its use in supporting repetitive work procedures are the reasons for high attractiveness to many lab users. Our initial results applying this industry-like assay on drug resistance testing on cell models are very encouraging. However, conclusive evaluations on whether data quality is improved requires further projects comparing manual pipetting-derived data with corresponding data retrieved from the robot tool. Such a project is currently underway.\n\n\nSummary\n\nWe present a rapid-to-implement, feedback approved method guide for initial steps to improve value of preclinical lab deliverables in a budget-restricted academic research environment. Given the increasing concerns on the value of preclinical research, we believe our activities are in line with current goals of funding authorities, academic self-governance and help to improve trust and recognition of science in society. We hypothesize that side effects of a QMS can also reduce inequality/discrimination amongst stakeholders.\n\n\nData availability\n\nZenodo: STR Analysis results BTSC 349, BTSC268 and mix of both. http://doi.org/10.5281/zenodo.390144612\n\nThis project contains the following underlying data:\n\n- 15-1-Wiss2020-01-16.fsa (BTSC 349 short tandem repeat analysis)\n\n- 15-3-Wiss2020-01-16.fsa (BTSC268 short tandem repeat analysis)\n\n- 15-4-Wiss2020-01-16.fsa (Mixed cell line short tandem repeat analysis)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nDIN EN ISO 9001: 2015-11 - Quality management systems. Beuth, 2015. Reference Source\n\nTackett JL, Miller JD: Introduction to the Special Section on Increasing Replicability, Transparency, and Openness in Clinical Psychology. J Abnorm Psychol. 2019; 128(6): 487–492. PubMed Abstract \| Publisher Full Text\n\nMiyakawa T: No raw data, no science: another possible source of the reproducibility crisis. Mol Brain. 2020; 13(1): 24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBaker M: Is there a reproducibility crisis? Nature. 2016; 533(7604): 452–454. Reference Source\n\nelabftw.net. [cited 2020 Mar 19]. Reference Source\n\neLabFTW version 3.4.8. [cited 2020 May 06]. Reference Source\n\nVargas-Toscano A, Khan D, Nickel AC, et al.: Robot technology identifies a Parkinsonian therapeutics repurpose to target stem cells of glioblastoma. CNS Oncol. 2020. PubMed Abstract \| Publisher Full Text\n\nVenkataramani V, Tanev DI, Strahle C, et al.: Glutamatergic Synaptic Input to Glioma Cells Drives Brain Tumour Progression. Nature. 2019; 573(7775): 532–538. PubMed Abstract \| Publisher Full Text\n\nWilkinson M, Dumontier M, Aalbersberg IJJ, et al.: The FAIR Guiding Principles for scientific data management and stewardship. Sci Data. 2016; 3: 160018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoher D, Bouter L, Kleinert S, et al.: The Hong Kong Principles for Assessing Researchers: Fostering Research Integrity. OSF. Reprints. 2019. Publisher Full Text\n\nGerman Research Foundation Guidelines for Safeguarding Good Research Practice, Code of Conduct. Self-published, 2019. Reference Source\n\nNickel AC: STR Analysis results BTSC 349, BTSC268 and mix of both. [Data set]. F1000Research. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3901446" }	[ { "id": "65819", "date": "17 Jul 2020", "name": "Jan Vollert", "expertise": [ "Reviewer Expertise Pain Research", "Placebo", "Improvement of Clinical and Preclinical Trial 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\nThe authors present the quality management system they have implemented in their lab and lay out specifics and improvements to their previous processes. The reader would benefit from more elaboration of the motivation that initiated the change, a better structure in before/after comparisons, and a primer in quantifying compliance and satisfaction of the users.\nSpecific points:\n“However, it is shown, that a lack of quality management and transparency are two reasons for the ongoing reproducibility crisis2–4. “\nWhile I fully agree with this statement, I think the reader would benefit from some elaboration on this topic: why and how do QS and in particularly organised documentation, increase reproducibility?\neLabFTW: it would be interesting to lead the reader through your decision process, as I assume other labs will consider similar options as you did. Why did you choose this one over the others?\n\nThe central element of this manuscript – the reporting on some bits on the motivation of the researchers to finally approach this step would be interesting, which could also allow for a better before/after comparison. Which processes have improved by the introduction of the new system, where do the users experience early benefit, where are the users rather reluctant?\n\nI appreciate that some information on the before is given in the results section, but think that a separate, earlier header on “before” would improve the structure.\nSimilarly, the reporting of enthusiasm within the group is relatively vague – maybe you could even add a small anonymous survey within the group which bits are most and least liked. This can quantify the impact of the system at least a bit.\n\nMinor point: I am always at risk of doing this myself, but I would recommend refraining from using terms like “significant influence” as the word “significance” is so strongly tied to p-values in research papers.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5779", "date": "06 Aug 2020", "name": "Michael Hewera", "role": "Author Response", "response": "Dear Jan Vollert,Thank you a lot for your very quick response!Following your advise, we added a short part about the process of choosing eLabFTW as out electronic lab book and changed the several \"significants\".We really liked your idea of small surveys, but think it would have been only feasable, if we also did the same one before the installation of the QMS.Inside of the team we do have our feedback meetings, to get these responses. Unfortunately we cannot publish our meeting minutes as data though.Best regards,Michael Hewera" } ] }, { "id": "65763", "date": "27 Jul 2020", "name": "Judith J. de Haan", "expertise": [ "Reviewer Expertise Human cellular immunology", "virology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is an interesting manuscript that shows that research groups can improve the quality of their experimental work without spending lots of money. It is great that researchers feel the need to improve their own practice, even if that does not immediately lead to more publications or more funding, but because it will improve the reproducibility of their work and contribute to the quality of the whole research field they are working in.\n\nSome suggestions for improvement:\n\nThe level of details in this paper vary a lot, on the one hand it is very detailed (the amount of DNA and volume for the reaction to check cell line stability), but the level of information on the bio bank is very limited.\n\nIt seems that the authors try to write a ‘case report’ about their own lab and at the same time try to give recommendations for other labs. It might be more valuable to make a clear distinction between those two goals. Maybe write it as a case report and then provide recommendations in the discussion part. Maybe distinguish the recommendations too, material wise recommendations (check cell lines, buy a pipet pump of ~1000 USD), (data ) storages recommendations and attitude recommendations (have regular lab meetings about quality, make sure all lab members use the same digital notebook and SOPs).\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5780", "date": "06 Aug 2020", "name": "Michael Hewera", "role": "Author Response", "response": "Dear Judith de Haan and Frank Miedema,Thank you for reviewing our publication.Following your suggestion, we adapted the level of detail, by adding some information to the bio bank part of the text.Best regards,Michael Hewera" } ] }, { "id": "65762", "date": "27 Jul 2020", "name": "Clarissa Carneiro", "expertise": [ "Reviewer Expertise Metascience and behavioral neuroscience." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary:\nThe article describes an experience with a quality management system (QMS) within an academic basic biomedical research laboratory. The subject is relevant to the life sciences in general, as concerns with research quality are on the rise, and describing the development and implementation of a QMS within a lab is a worthy contribution; however, we believe the article would benefit from major changes in its structure to make its objectives, methods and results clearer.\nMain concerns:\nWho is the intended audience for the article? If this is meant to be a template or example to be followed by laboratory researchers in general, we believe that more information about the concept of QMS and the process of developing it are probably warranted. On the other hand, very specific information about cell culture automation and STR profiling, such as that presented on Table 1, is only of interested to a very specific audience. If the article aims to be a description of QMS in cell culture labs, that would be a valid goal as well, but in this case this topic should be covered in more detail, and the text in other sections should be changed in order to reflect this aim.\n\nWhat are the objectives of the article? If the goal is to describe the effects of the QMS system on some aspects of quality, more data is needed to adequately assess its impact. If the goal of the article is to present the development of the QMS, that is fine as well, but the methods and results sections should then be restructured to reflect that goal – in this case, the methods should explain the decision process leading to QMS implementation, while the results would present the system as it was implemented. In either case, a more detailed description of each block of the QMS is warranted, either as methods or as results, depending on the objective of the article.\n\nWhat is meant by quality and by reproducibility? Both terms can be read with many different interpretations, so including definitions would help to avoid misinterpretations of the goals of the article. Note that the QMS seems to address multiple facets of these concepts, so clarification might be necessary within each block of the QMS. In particular, most lab researchers are not familiar with the literature on quality management, so a more in-depth review of these concepts is needed in the introduction.\nTitle and abstract:\nGiven the direct and practical content of the article, we suggest the title would be improved by removing the first part (i.e. “Measures to increase value of preclinical research”) and leaving only the second one.\n\nWhile the abbreviation QMS is defined in the abstract, we believe it should not be used in the title, in which it cannot be defined. Instead, we suggest using the unabbreviated form.\n\nThe whole abstract could be shortened and made more direct, particularly in the Introduction and Methods section.\n\nIf the system has already been implemented (as seems to be the case), why are some sentences in the future in the methods section of the abstract (e.g “These will be stored…”)?\n\nThe abstract “Methods” subsection contains information not available elsewhere in the full text article (i.e. “Moreover, we restrict presentations of our actions on those for what we received a positive feedback by the users regarding its applicability”). Moreover, such feedback data is never presented in the article.\n\nThe “Results” subsection does not seem to contain any result, irrespective of what objective is intended (as described above).\nIntroduction:\nIf the article is targeted at a life sciences audience, however general or specific, we would not expect most of them to be familiarized with quality assessment vocabulary or literature. With that in mind, we suggest including an operational definition of QMS. Additionally, there has been some debate about its implementation in the basic academic research lab; thus, describing this context and reviewing the literature on the subject would also beneficial to the readers.\n\nOn a more specific development of the previous point, we suggest including a brief description of your motivations for engaging in this endeavor. Was this a requirement by a superior instance, such as the institution or a funding agency? Was this a bottom-up initiative from the researchers? This could both (a) help to reach readers that could identify with the same issues but were not aware of them and (b) help to contextualize the adopted strategies in a broader landscape of previous experiences.\n\nIndependently of the goal or targeted audience, for a case-study such as this one, it is vital that the introduction includes a description of the lab setting for which this QMS was developed. This includes the number of people and their positions in the lab (such as students, technicians, postdocs, etc), the group’s research interests or focus areas and the institutional environment (such as governmental vs. private funding, discovery-based vs. applied, etc). While some of these features can be inferred from other sections of the text, they should be presented upfront in the Introduction.\n\nThe three references cited on “that a lack of quality management and transparency are two reasons for the ongoing reproducibility crisis” do not seem to fully support the sentence, as they do not directly mention quality management. That sentence should either be corrected or clarified to better represent the cited articles.\nMethods:\nThe issues raised as general comments should have a strong impact on the structure of the methods section. If the goal is to describe the creation of the QMS, the decision process to create each component of the system should be included here. If the goal was to assess the QMS itself, a more objective description of the QMS itself is warranted, but new sections on data collection would be required. In each subsection of the Methods, we found one or a combination of these strategies, which causes the section to lack coherence and makes the reader unsure about what the objectives actually were.\n\nFrom our understanding, Figure 1 is intended to be a summary of the QMS system. However, it does not completely correspond to what is described in the text. On one hand, the “lab meeting deciding core rules of QMS” shown in the first block of the figure is not described in the Methods section; on the other the “Quality of Cell Cultures” subsection is not reflected in the figure.\n\nIn the description of standard operating procedures (SOPs), it is not clear for what processes they are being developed. Listing at least the most used SOPs would greatly improve our understanding of the strategy; one cannot understand for example, how specific a process is described in each of the 91 SOPs. Ideally, the authors should share some examples in the article, and perhaps include a template as a supplementary material.\n\nThe description of one-to-one meetings probably deserves its own subheading and should not be under the SOP subsection.\n\nAs mentioned previously, the quality of cell culture assessments subsection seems out of context and should only be included if the authors mean to reach a very specific audience (e.g. labs mainly working with cell cultures). If they mean to use cell culture as an example of the QMS implementation, we would suggest describing it in the Results section and relating each step of the process to the QMS blocks that are described in the methods.\n\nIn the Feedback and Update section, there are three consecutive sentences with “hypothesize”, “believe” and “think” as verbs. That said, do the authors have data on the subjects touched upon? If so, they should present it – otherwise, they might be excessively speculating on the impact of their intervention.\n\nAlso in this section, the link between the meetings and the claims of reduced inequality and discrimination is not clear. Please clarify.\n\nWhen describing automation, a result is mentioned: “i.e. the application of the screening identified a repurpose of a FDA approved neurotransmitter drug to inhibit growth of brain tumor cells…”. This does not belong in the Methods section and should be moved to the Results. Moreover, the causal link between automation and the mentioned results is not obvious and could be better explained by the authors.\nResults and discussion:\nAs mentioned above, the structural questions about the objective of the paper addressed in the General section of this report should have an important impact on the “Results and discussion” section of the article. In general, we found it to be better structured than the “Methods” section. In particular, we appreciated the description of some quantitative results and recognition of some limitations. That said, the section is still scarce on providing actual measures of impact of the QMS implementation – and, if this is meant to be the goal of the article, should include more data on the subject.\n\nIn the first paragraph, the authors mention an increase in team motivation and improvement of scientific development of lab members, while the second paragraph states that “now a higher consistency of experimental results is given”. Do the authors have any data to base these statements on? If so, it should be shown (and details of this assessment should be included in the methods). If not, these conclusions might not be warranted.\n\nWe suggest including more information when describing the implementation of the electronic laboratory notebook. For example, this particular product requires server installation, which might not be accessible to many biomedical researchers. Did you have assistance from an IT department of your institution? Moreover, how was the process for the 13 people who were able to completely migrate to this system? Did they have to stop their experimental schedules to implement all changes at once or was it gradual?\n\nThe description of cell culture contaminations (Table 1) is very specific and not understandable for readers with no experience with STR profiling. If this is to be presented, it definitely needs a general description of how the results should be read and interpreted. That said, perhaps the best option would to leave this information out of the main text of the article and present it as supplementary material.\n\nTo support the claims of this being a QMS for resource-restricted research groups, more information regarding the implementation cost of all blocks of the system is needed. Exemplifying the financial costs of the cell culture assessment assays is a welcome contribution; this, however, could be expanded in other parts of the QMS, not only in terms of financial costs but also of human resources and time requirement.\n\nIn various points, but especially when discussing meetings (i.e. “a non-scientific oriented lab meeting creates an opportunity for easier expression of general matters…”), it would be useful to know whether the authors’ impression is indeed shared by all of the lab personnel. Do they have any kind of questionnaire/feedback data to know whether this opinion extends beyond the authors themselves?\nSummary:\nWe suggest toning down the conclusions presented here. Given the methods and results presented as they are, there is little data to support the claims of fast, easy or low-cost implementation of the QMS. To substantiate this claim, more details relating to cost, speed and implementation challenges should be provided in the previous section.\n\nThe same recommendation holds for increased value of preclinical lab deliverables – unless the authors present more data on the impact of QMS implementation, this conclusion might not be warranted.\n\nOnce more, it is not clear what the hypothesis of a reduction of inequalities (also mentioned in the methods) and discrimination is based on, as this does not seem to follow logically from QMS implementation.\nReferences:\nIn general, we do not believe the study provides an adequate reference list. It is insufficient to provide context to the methods used or to the discussion presented.\n\nReference 4 has the wrong title and links to an unofficial source. It should be corrected to the publisher’s version.\n\nThere is a typo in Reference 10: it should read OSF Preprints.\n\nReference 12 is listed with two “publishers”: F1000 and Zenodo. From the link, we infer that Zenodo is the correct one.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5783", "date": "06 Aug 2020", "name": "Michael Hewera", "role": "Author Response", "response": "Dear Clarissa Carneiro and Olavo B Amaral,We thank you a lot for your very detailed review of our publication.Most of your suggestions have been introduced in the revised version of the article.Best regards,Michael Hewera" } ] } ]	1	https://f1000research.com/articles/9-660
https://f1000research.com/articles/9-914/v1	05 Aug 20	{ "type": "Research Article", "title": "Poor eating habits and predictors of weight gain during the COVID-19 quarantine measures in Kuwait: a cross sectional study", "authors": [ "Nouf AlMughamis", "Shaimaa AlAsfour", "Shariq Mehmood", "Shaimaa AlAsfour", "Shariq Mehmood" ], "abstract": "Background: Despite the public health importance of documenting the burden of physical inactivity and weight gain, there is a paucity of such data in Kuwait during the lockdown for Covid-19 pandemic. Therefore, this survey was designed to estimate: the burden of poor eating habits particularly binge eating habits, fluctuations in weight and its predictors among the Kuwaiti public. Methods: This cross-sectional survey was conducted from 2nd to 12th April 2020 among the general public in Kuwait. All data were collected through a social media platform (WhatsApp groups), through convenience and snowball sampling methods. The survey comprised three sections: a) demographic characteristics of respondents, b) eating habits particularly binge eating, consumption of snacks and beverages c) subjective feelings of anxiety and d) weight before and during the pandemic. All data were analyzed using SPSS (v.25). Results: There was a total of 522 valid respondents, with a mean age of 41.78 (11.75) years. There was a significant increase in weight of respondents during the quarantine (mean difference= -1.13, SD 5.39, t= -4.52, p < 0.001). Those with reporting unhealthy diets were 4.5 times more likely to report an increase in weight. Those reporting having anxiety throughout the day were 2.45 times more likely and those consuming snacks excessively were associated with 3.27 times higher odds of increase in weight than those not consuming it. Conclusions: We recommend that authorities develop a strategic plan to counter harmful effects of this pandemic on health that are originating from sedentary lifestyle, unhealthy eating patterns and psychological issues.", "keywords": [ "weight gain", "COVID-19", "quarantine", "binge-eating", "anxiety", "Kuwait" ], "content": "Introduction\n\nThese are unusual times, as the coronavirus disease 2019 (COVID-19) pandemic of has drastically affected daily lives of millions of people around the world. The concept of quarantine is practically new for the general public, and has influenced social, economic and psychological threads of our life1,2. Due to changes in our habits including socializing, physical work, eating and entertainment, it is natural that level of anxiety and depression is on the rise among people in quarantine3. The psychological impact of quarantine has also been associated with post-traumatic stress, confusion and aggression1.\n\nAmong the general public, poor information pertaining to the COVID-19 and the rationale for lockdown has given birth to fear toward infection, frustration, boredom, financial loss and stigma1. These stressors may lead to people being homebound and lead a sedentary lifestyle and develop poor eating habits. This poor lifestyle can be detrimental to the health structure of Middle Eastern countries, especially Kuwait, where obesity has a high prevalence and a large proportion of Kuwaiti national meet the World Health Organization’s definition of ‘obesity’4. According to Al-Nohair, the prevalence of obesity among Kuwaiti males is 36% and in females is 48%4,5. This high rate of obesity in Kuwait has been attributed to a plethora of biopsychosocial risk factors and a polygenic etiology4. The current pandemic and associated quarantine may further exacerbate the situation1.\n\nDue to the disrupted daily routine of adults and children, less social contact and rising psychological distress, the health of Kuwaiti residents can further deteriorate both physically and mentally1. All of these factors have been documented to particularly affect healthy eating habits6. Over-eating and the resulting weight gain during the COVID-19 quarantine, may result in an increase in incidence of chronic diseases such as diabetes, cardiovascular diseases and psychiatric illnesses. Epidemiological and public health studies focusing on nutrition and weight gain among the general masses are needed to develop strategies and devise interventions countering these.\n\nDespite the public health importance of documenting the burden of physical inactivity and weight gain, there is a paucity of such data in Kuwait. Therefore, this survey was designed to estimate the burden of poor eating habits particularly binge eating habits, fluctuations in weight and its predictors among the Kuwaiti public.\n\n\nMethods\n\nThis cross-sectional survey was conducted from 2nd to 12th April 2020 among the general public in Kuwait City, Kuwait. The country reported its first case on 24th February 2020, and the country went into partial lockdown since 22nd March, and on 10th May, the country was placed in full curfew based on Ministry of Health’s assessment7 (at the time of writing, on 4th August 2020 Kuwait will remain under curfew 9pm-3am). All data were collected through a social media platform (WhatsApp groups), where first participants were chosen through convenience sampling and the rest through snowball sampling methods. Attempts were made to exclude non-Kuwaiti nationals by distributing the questionnaire to Kuwaiti nationals and screening the datasets after collection of data. Inclusion criteria included participants being Kuwaiti nationals residing in Kuwait. There were no specific exclusion criteria pertaining to age, language, gender or occupation of the respondents. After providing written informed consent, the respondents were briefed about the objectives of the survey, ensured anonymity and that the group findings will be reported. Respondents could leave the study at any time if they felt uncomfortable. Approval for this study was provided by the Ethical Review Board of Ministry of Health of Kuwait, Kuwait (Approval #2020/1477).\n\nThe minimum sample size required for this survey was calculated to answer out primary research objective of documenting prevalence of poor eating habits among Kuwaitis. Based on a statistical power of 80%, confidence level 95%, margin of error 5% and a population size of 1.3 million Kuwaitis, a minimum sample size of 385 was required. All data were collected using an electronic version of a bilingual questionnaire in Arabic and English languages. The questionnaire was pre-piloted among a few participants before distribution; however, no factor validity or reliability testing was done due to categorical and open-ended nature of the responses. Participants were encouraged to provide their responses in either of the languages. The questionnaire comprised of three sections20: a) demographic characteristics of respondents, b) eating habits particularly binge eating, consumption of snacks and beverages c) subjective feelings of anxiety and d) weight before and during the pandemic. Demographic characteristics included gender, age, nationality, marital status, parity and housing of the respondents. Several questions were asked regarding binge eating habits documenting change in nutrition habits, time of increased food consumption, number and type of snack foods consumed during the day, water and coffee intake. Physical activity during the day was inquired using two questions: a) a trichotomous question documenting change in physical activity when compared with normal routine before the quarantine and number of hours spent in a sedentary manner per day in the last week. Symptoms of anxiety were measured subjectively using a three-point Likert scale. Participants were asked the extent to which they experienced anxiety every day in last 7 days. Responses were given on a three point scale ranging from never to always. Participants were instructed to respond with their weights measured using a weight scale (digital or analogue) if they had measured it. Similarly, they were instructed to input their current weight status using a digital or analogue weighing scale, if available. The question regarding weight of the respondents before and during the lockdown, yielded 462 (88.50%) valid responses. The remaining participants could not input their weights due to unavailability of weighing scales.\n\nAll data were analyzed using SPSS (v25). Descriptive statistics were calculated for all variables where quantitative variables were presented as mean (SD) and categorical ones as frequency and percentage. Statistical significance for difference between weight of respondents during the COVID-19 pandemic and before it was assessed using t-test for dependent samples. To delineate predictors of weight gain, it was planned a priori to run multiple regression; however, due to the non-normalized nature of the data, logistic regression analysis was performed.\n\nThereafter, the respondents were categorized in to two groups: participants with weight gain and participants with negative or no change in weight. To identify the predictors of experience in weight gain among the Kuwaitis, binary logistic regression (stepwise method) was employed. Theoretically sound factors showing relationship with weight gain in previous literature were added as predictors in the logistic regression model8. These predictors included demographic characteristics of respondents, eating habits c) subjective feelings of anxiety and d) questions pertaining to physical inactivity. The final model reported significant predictors of weight, with statistical significance set at ≤0.05 and the variance in the model was assessed using the Negelkerke R2.\n\n\nResults\n\nThere was a total of 522 valid respondents, with a mean age of 41.78 (11.75) years. More of the respondents were females (380, 72.8%) than males (142, 27.2%). A higher proportion of the respondents were married (379, 72.6%) than single (97, 18.6%). A total of 201 (38.5%) respondents reported having 1–3 children, followed by >3–6 (170, 32.6%), no children (124, 23.8%) and more than 6 children 27 (5.2%). More than half of the respondents lived in their own houses 297 (56.9%), followed by at parents’ house 128 (24.5%), and apartment 97 (18.6%).\n\nA higher proportion of the respondents’ families were assisted by a cook (299, 57.3%) to prepare meals than themselves (223, 42.7%). A change in nutritional habits was reported by 387 (74.1%) respondents, with 214 reporting that their eating pattern had improved to a healthier one (214, 41%) than unhealthy one (24.3%). Respondents tend to consume more food at night (232, 44.4%) than during daytime. A total of 352 respondents (67.4%) reported consuming snacks more than twice per day. Water intake was self-reported as enough (287, 55.0%) and 300 (57.5%) respondents reported having 1-3 cups of coffee a day.\n\nFor the question regarding weight of the respondents before and during the lockdown, there were 462 (88.50%) valid responses. We did not impute the missing data for responses regarding weight measurements, and excluded participants with missing data in the logistic regression model. Mean weight of respondents before the pandemic was 75.03 (16.85) kg and 75.74 (17.54) kg during the pandemic (Figure 1). There was a significant increase in weight of respondents during the quarantine (mean difference= -1.13, SD 5.39, t= -4.52, p < 0.001). A higher proportion of respondents reported that their weight will stabilize after the pandemic (n=305, 58.4%) while 217 (41.6%) reported that it would increase. A total of 333 (63.8%) of the respondents reported feeling anxious sometimes during the day, 133 (25.5%) always and 56 (10.7%) never. Over 69% of the respondents reported that their physical activity had reduced than before. Mean number of hours spent being sedentary at home were 9.56 (5.64) (Table 1).\n\nLogistic regression (stepwise method) was used to analyze statistically significant predictors of increase in weight among the Kuwaitis during the COVID-19 quarantine. It yielded a significant model (X2= 90.43, df= 8, p< 0.001). The model explained 24.9% of variance in weight changes among Kuwaitis (Nagelkerke R2) and correctly classified 71.6% of the cases, whereas the sensitivity of this particular model was 65.8% and specificity 78% in predicting the increase in weight among Kuwaiti respondents. According to this model, when compared with respondents who changed their diet pattern to healthier, those with reporting unhealthy diets were 4.5 times (95% CI= 2.45 to 8.23) more likely to report an increase in weight. Those reporting having anxiety throughout the day were 2.45 times more likely to have an increase in weight than those never experiencing it. Consuming snacks excessively\n\n(>3 times a day) was associated with 3.27 times higher odds of increase in weight than those not consuming it. Those that consumed moderate amounts of snacks (1–3 times a day) did not differ to their counterparts who never consumed snacks throughout the day (Table 2).\n\n\nDiscussion\n\nThe present study documents change in dietary patterns among Kuwait residents during the COVID-19 pandemic. According to our results, a high proportion of Kuwaitis reported binge eating habits, a sedentary lifestyle and an increase in their weight during the quarantine. Significant predictors of weight gain among the Kuwaitis included unhealthy diet pattern, excessive consumption of snacks and subjective feelings of anxiety.\n\nOur study reports a high prevalence of unhealthy eating habits and sedentary lifestyle during the pandemic, which contributed to a significant weight increase among the Kuwaitis. Although in the past, several studies have reported high rates of unhealthy habits and obesity among the Kuwaiti public, this situation may have worsened during the lockdown. This time of pandemic and quarantine has affected almost all aspects of life and the pattern of unhealthy eating habits have become more prevalent including excessive consumption of snacks, predicting an increase in weight of the studied population. Our study also showed that a higher proportion of respondents assume that their eating pattern has become healthier but at the same time the frequency of snacks binge eating, and night-time consumption of food showed statistical significance, leading to unhealthy patterns of food consumption. An increase in weight gain and development of unhealthy eating habits will present as a challenge in the post-COVID era, and policies must be devised to strengthen health systems to tackle this.\n\nAnother important predictor of weight included subjective feelings of anxiety during this era, which not only carry negative physical and mental health implication but also contribute to ‘stress-eating’ and ‘food cravings’9–14. Feeling anxious during this era of pandemic is natural and eating more carbohydrates and binge eating likely improves mood in the short term, but leads to gain of weight and chronic diseases incidence also contribute to poorer body image9–14. This double burden of poor mental health and obesity thus presents a great challenge to the public health professionals and interventions must be devised to ensure good mental health during these trying times. Several interventions, especially psychoeducation related to this pandemic and meditation interventions, can be provided to ease off worry and stress and reduce anxiety levels among the general public.\n\nThe results from this study can serve as a guiding tool for shaping health strategy during this pandemic by health authorities. As Gulf countries grew enormously in wealth, living standards were raised, albeit at a cost of more sedentary and comfortable life routine5,15,16. Kuwait had previously developed a ‘Kuwait National Programme for Healthy Living (2013-2017)’17; authorities can develop a strategic plan to counter harmful effects of this pandemic on health that are originating from sedentary lifestyle, unhealthy eating patterns and psychological issues.\n\nDespite its large sample size, this study has several limitations which need to be considered for careful interpretation of its results. Most important limitations include a convenience and snowballing approach for collection of data as opposed to random sampling. Cross-sectional nature of this study limits inferences related to causality and temporality and use of a subjective question for estimating anxiety levels limit validity of these results and also introduce recall bias. Future studies should consider a prospective study design and use of valid and reliable questionnaires like the Generalized Anxiety Disorder 7-item scale18.\n\n\nData availability\n\nFigshare: Diet Kuwait.csv, https://doi.org/10.6084/m9.figshare.12706223.v119.\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: CovidKuwaitWt-Survey.pdf, https://doi.org/10.6084/m9.figshare.12709631.v120.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe authors thank Dr. Ahmed Waqas, doctoral fellow at the University of Liverpool, Liverpool, UK, for providing assistance in data analysis.\n\n\nReferences\n\nBrooks SK, Webster RK, Smith LE, et al.: The psychological impact of quarantine and how to reduce it: rapid review of the evidence. Lancet. 2020; 395(10227): 912–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaplan J, Frias L, McFall-Johnsen M: A third of the global population is on coronavirus lockdown — here’s our constantly updated list of countries locking down and opening up. 2020. Reference Source\n\nZohn Rosen SA, Cheskie Rosenzweig WL, Rosmarin DH, et al.: Anxiety and distress among the first community quarantined in the U.S due to COVID-19: Psychological implications for the unfolding crisis. J Chem Inf Model. 2020; 53: 1689–99. Publisher Full Text\n\nAlnohair S: Obesity in Gulf Countries. Int J Health Sci (Qassim). 2014; 8(1): 79–83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBadr HE: Differences in physical activity, eating habits and risk of obesity among Kuwaiti adolescent boys and girls : a population-based study. 2017; 1–9. Publisher Full Text\n\nMuscogiuri G, Barrea L, Savastano S, et al.: Nutritional recommendations for CoVID-19 quarantine. Eur J Clin Nutr. 2020; 74(6): 850–851. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nState of kuwait: COVID-19 updates. 2020. Reference Source\n\nZachary Z, Brianna F, Brianna L, et al.: Self-quarantine and weight gain related risk factors during the COVID-19 pandemic. Obes Res Clin Pract. 2020; 14(3): 210–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVidovi V, Henigsberg N, Jure V: Anxiety and Defense Styles in Eating Disorders. 2003; 1: 125–34. Reference Source\n\nAoun C, Nassar L, Soumi S, et al.: The Cognitive, Behavioral, and Emotional Aspects of Eating Habits and Association With Impulsivity, Chronotype, Anxiety, and Depression: A Cross-Sectional Study. Front Behav Neurosci. 2019; 13: 204. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHiggins DM, Dorflinger L, MacGregor KL, et al.: Binge eating behavior among a national sample of overweight and obese veterans. Obesity (Silver Spring). 2013; 21(5): 900–3. PubMed Abstract \| Publisher Full Text\n\nVerplanken B: Beyond frequency: Habit as mental construct. Br J Soc Psychol. 2006; 45(Pt 3): 639–56. PubMed Abstract \| Publisher Full Text\n\nYannakoulia M, Panagiotakos DB, Pitsavos C, et al.: Eating habits in relations to anxiety symptoms among apparently healthy adults. A pattern analysis from the ATTICA Study. Appetite. 2008; 51(3): 519–25. PubMed Abstract \| Publisher Full Text\n\nSchulz S, Laessle RG: Associations of negative affect and eating behaviour in obese women with and without binge eating disorder. Eat Weight Disord. 2010; 15(4): e287–93. PubMed Abstract \| Publisher Full Text\n\nMusaiger AO: Consumption, Health Attitudes and Perception Toward Fast Food Among Arab Consumers in Kuwait: Gender Differences. Glob J Health Sci. 2014; 6(6): 136–43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBalhareth A, Meertens R, Kremers S, et al.: Overweight and obesity among adults in the Gulf States: A systematic literature review of correlates of weight, weight-related behaviours, and interventions. Obes Rev. 2019; 20(5): 763–93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBehbehani K: Kuwait National Programme for Healthy Living : First 5-Year Plan ( 2013 – 2017 ). Med Princ Pract. 2017; 23 suppl 1(Suppl 1): 32–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSpitzer RL, Kroenke K, Williams JBW, et al.: A brief measure for assessing generalized anxiety disorder: The GAD-7. Arch Intern Med. 2006; 166(10): 1092–7. PubMed Abstract \| Publisher Full Text\n\nAl Mughaimis N: Diet Kuwait.csv. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12706223.v1\n\nAlMughamis N: CovidKuwaitWt-Survey.pdf. figshare. Preprint. 2020. http://www.doi.org/10.6084/m9.figshare.12709631.v1" }	[ { "id": "79339", "date": "04 Mar 2021", "name": "Maha AlHussain", "expertise": [ "Reviewer Expertise Meal pattern", "obesity", "Energy balance." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 was estimating the burden of poor eating habits, fluctuations in weight and its predictors among 522 Kuwaiti public. The main finding is that people with reporting unhealthy diets were 4.5 times more likely to report an increase in weight. There are several comments/suggestions that must improve this work:\n\nInadequate methods description. For example, does the study sample include children? If so, was the informed consent provided from parents/guardians? The authors mentioned that attempts were made to exclude non-Kuwaiti nationals. It is not clear whether all non-Kuwaiti national have been excluded or not.\n\nData were analysed by SPSS, but were the data cleaned? What cleaning was done, should be included.\n\nWere the numerical data tested for normality?\n\nWhat was the response rate?\n\nSelf-assessment body weight measures should acknowledge as a limitation.\n\nThe conclusion is inadequate and need to be expanded according to the study results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "80146", "date": "08 Mar 2021", "name": "Gorica Maric", "expertise": [ "Reviewer Expertise Epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFirst, I would like to thank the Editorial Team for providing me chance to review an article for F1000 Research. The manuscript entitled \"Poor eating habits and predictors of weight gain during the COVID-19 quarantine measures in Kuwait: a cross sectional study“ presents data on eating habits and changes in body weight along with its predictors in the population of Kuwait city. Although the topic is very actual and important from many aspects, there are some methodological issues that need to be clarified:\nUsing Whatsapp groups for reaching participants in my opinion represents significant sampling bias, maybe some people don’t use this app, other (elderly) are not so familiar with its use, etc.\n\nThe questionnaire: The data on the questionnaire development is scarce. Did the authors use some pre-existing questionnaire or developed the new one? If so, how did they make it, based on which sources? What were the results of pre-pilot testing, did the questionnaire undergo any changes after that?\n\nHow many people were invited for the participation in the survey, and how many of them accepted and how many refused participation in the study?\n\nPlease, add the age distribution by categories as well.\n\nAre the age and gender distribution in study sample in accordance with the age and gender of Kuwait city population?\n\nSelf-measurement of the body weight should also be stated as a study limitation.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-914
https://f1000research.com/articles/9-903/v1	04 Aug 20	{ "type": "Brief Report", "title": "Genome-wide analyses reveal antibiotic resistance genes and mechanisms in pathogenic Pseudomonas bacteria", "authors": [ "Otarigho Benson" ], "abstract": "Background: The global emergence and re-emergence of antibiotic resistance among the Pseudomonas pathogens causes great problems to patients undergoing chemotherapy. However, there is limited comparative information on the antibiotic resistance genes (ARGs) and mechanisms across the Pseudomonas pathogenic groups. Methods: The complete genomes of five Pseudomonas pathogen groups, P. aeruginosa, P. fluorescens, P. putida, P. stutzeri and P. syringae, were analyzed for ARGs. Results: A significant number of ARGs were identified in the P. aeruginosa genome compared to the other Pseudomonas pathogens. The opportunistic pathogens P. stutzeri and P. putida were shown to be the closest to P. aeruginosa with an average nucleotide identity (%) of 80.30 and 79.52. The pathogen genome with the least hit was P. stutzeri. The four major antibiotic resistance mechanisms that include the efflux, inactivation, target alteration and efflux::target alteration were reported. Conclusion: The findings of this brief report could be useful in understanding the chemotherapeutics against antibiotic resistance strains of Pseudomonas pathogens", "keywords": [ "Pseudomonas", "Antibiotics Resistance", "bacterial pathogen", "P. aeruginosa" ], "content": "Introduction\n\nThe emergence of Gram-negative bacterial antibiotics resistance is a growing threat to antibiotic therapy. The bacterial pathogens in the genus Pseudomonas are mostly opportunistic that cause great damage and loss of life (Breidenstein et al., 2011; Evans et al., 2008; Kawai, 1974; Lalucat et al., 2006). These Pseudomonas pathogens are pervasive that is able to infect, survive and proliferate in a wide range of biotic and abiotic environments (Azam & Khan, 2019; Silby et al., 2011). Presently, there are seven groups in the genus Pseudomonas, with P. aeruginosa being the most pathogenic (Barbier et al., 2013) and which causes high morbidity and mortality in cystic fibrosis patients and immunocompromised individuals (Sadikot et al., 2005). The other Pseudomonas pathogenic groups include P. fluorescens (Biaggini et al., 2015), P. putida (Fernández et al., 2015), P. stutzeri (Lalucat et al., 2006) and P. syringae (Xin et al., 2018). P. aeruginosa is the major cause of infections in developed countries due to its highly evolved resistance to a wide variety of antibiotics (Hancock & Speert, 2000) making it very difficult to treat and limiting therapeutics (Breidenstein et al., 2011). Even though P. aeruginosa is the most studied, there are some other Pseudomonas species that exhibits opportunistic pathogenic behavior to animals (P. fluorescens, P. putida and P. stutzeri) (Azam & Khan, 2019) and plants (P. syringae) (Xin et al., 2018). Most of the known antimicrobial resistance (AMR) gene and mechanistic studies are focused on P. aeruginosa with little attention directed to other Pseudomonas pathogens. Hence, there is need to investigate different Pseudomonas pathogens genomes for diverse antibiotic resistance genes (ARGs) and mechanism. Therefore, this brief report concisely revealed the ARGs, mechanisms and drugs in P. aeruginosa in comparison to other Pseudomonas pathogens.\n\n\nMethods\n\nThe complete genomes of the five groups of Pseudomonas pathogens, which included the P. aeruginosa (NC_002516.2), P. fluorescens (NC_016830.1), P. putida (NC_002947.4), P. stutzeri (NC_015740.1) and P. syringae (NC_007005.1) fasta file sequences, were downloaded from The National Center for Biotechnology Information Genome database. The five Pseudomonas pathogen genomes were selected to represent the Pseudomonas groups. The fasta file format of the genome sequence of bacteria were thoroughly analyzed for ARGs on the bulk analysis Resistance Gene Identifier (RGI) 5.1.0, CARD 3.0.9 Platform (Alcock et al., 2020) to extract AMR Genes, AMR Gene Family, Drug Class and Resistance Mechanism data. Default select criteria, which identified gene based on strict or perfect only was used. On the RGI platform, each genome sequence file was uploaded and all settings were left at default. The resistance genes, mechanism and drugs obtained from RGI platform were further analyzed using Prism 8 for number of ARG hits, gene family, mechanism and drug class per Pseudomonas species.\n\n\nResults\n\nThe five Pseudomonas pathogens show significant genome similarity (Table 1). P. stutzeri and P. putida, which have been reported to be opportunist pathogens to humans, were shown to be the closest relations to P. aeruginosa, with an average nucleotide identity (%) of 80.30 and 79.52. P. syringae, which infects plants, had the lowest average nucleotide identity (%) of 78.67. A number of antimicrobial resistance hits and genes were identified across the five Pseudomonas pathogens, but the highest hits were seen in the P. aeruginosa genome. The pathogen genome with the least hit was P. stutzeri (Figure 1A, B). The four major antibiotic resistance mechanisms identified were the efflux, inactivation, target alteration and efflux::target alterations in the P. aeruginosa genome. The efflux, inactivation and efflux::target alterations were identified in the P. fluorescens genome, while only the efflux and inactivation alterations were identified in the P. putida, P. stutzeri and P. syringae genomes (Figure 2A). The number of drug classes that P. aeruginosa was shown to be resistant to is also shown in Figure 2B.\n\n(A) The number of hit per each Pseudomonas pathogen genome (B). Number of ARGs per each Pseudomonas pathogen genome.\n\n(A) Number of resistance mechanism per each Pseudomonas pathogen genome. (B) P. aeruginosa resistance drug class. Drug class key: A= aminoglycoside antibiotic; B= fluoroquinolone antibiotic, diaminopyrimidine antibiotic, phenicol antibiotic; C= macrolide antibiotic, carbapenem, tetracycline antibiotic, acridine dye, diaminopyrimidine antibiotic, phenicol antibiotic; D= macrolide antibiotic, fluoroquinolone antibiotic, aminoglycoside antibiotic, carbapenem, cephalosporin, cephamycin, penam, tetracycline antibiotic, acridine dye, phenicol antibiotic; E= macrolide antibiotic, fluoroquinolone antibiotic, aminoglycoside antibiotic, cephalosporin, penam, tetracycline antibiotic, aminocoumarin antibiotic, diaminopyrimidine antibiotic, phenicol antibiotic; F= macrolide antibiotic, fluoroquinolone antibiotic, cephalosporin, penam, tetracycline antibiotic, aminocoumarin antibiotic, diaminopyrimidine antibiotic, phenicol antibiotic; G= macrolide antibiotic, fluoroquinolone antibiotic, monobactam, carbapenem, cephalosporin, cephamycin, penam, tetracycline antibiotic, peptide antibiotic, aminocoumarin antibiotic, diaminopyrimidine antibiotic, sulfonamide antibiotic, phenicol antibiotic, penem; H= peptide antibiotic; I = phenicol antibiotic; J = sulfonamide antibiotic.\n\n\nDiscussion\n\nThere is an increasing interest and focus in the antibiotic resistance in pathogenic Pseudomonas (Blair et al., 2014; Blanco et al., 2016; Li et al., 2015; Soto, 2013; Webber & Piddock, 2003). Hence, this report took advantage of the available Pseudomonas pathogens genomes and analysed for antibiotic resistance genes and mechanisms against different available drugs.\n\nThe significant number of ARGs and mechanisms were identified in the genome of P. aeruginosa, which is more virulent and well-studied compared to other species. P. aeruginosa also infects a wide range of plants and animals, including humans (Azam & Khan, 2019; Hancock & Speert, 2000; Sadikot et al., 2005). P. aeruginosa is of great medical importance due to its exhibition of multidrug resistance and its association with serious illnesses (Breidenstein et al., 2011; Evans et al., 2008; Gonzalez et al., 2019).\n\nThe most common resistance mechanism of Pseudomonas pathogens is the antibiotic efflux pump mechanism. Although this mechanism is most often seen in P. aeruginosa, it is also found in other Pseudomonas pathogen genomes. It has long been known that the antibiotic efflux pump is a key mechanism of resistance in Gram-negative bacterial pathogens (Blair et al., 2014; Blanco et al., 2016; Soto, 2013; Webber & Piddock, 2003). An antibiotic resistance strain’s efflux pumps allow it to regulate itself by excluding toxic substances, including antimicrobial drugs (Blair et al., 2014; Li et al., 2015; Soto, 2013; Webber & Piddock, 2003).\n\n\nConclusion\n\nThe different ARGs and mechanisms against known drugs in P. aeruginosa in comparison to other Pseudomonas pathogen were investigated and concisely reported in this brief report. The findings in this report could be useful in understanding the use of chemotherapeutics against antibiotic-resistant strains of Pseudomonas pathogens.\n\n\nData availability\n\nTable 1 lists the NCBI Genome accession numbers used in this study.", "appendix": "References\n\nAlcock BP, Raphenya AR, Lau TT, et al.: CARD 2020: antibiotic resistome surveillance with the comprehensive antibiotic resistance database. Nucleic Acids Res. 2020; 48(D1): D517–D525. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAzam MW, Khan AU: Updates on the pathogenicity status of Pseudomonas aeruginosa. Drug Discov Today. 2019; 24(1): 350–359. PubMed Abstract \| Publisher Full Text\n\nBarbier F, Andremont A, Wolff M, et al.: Hospital-acquired pneumonia and ventilator-associated pneumonia: recent advances in epidemiology and management. Curr Opin Pulm Med. 2013; 19(3): 216–228. PubMed Abstract \| Publisher Full Text\n\nBiaggini K, Barbey C, Borrel V, et al.: The pathogenic potential of Pseudomonas fluorescens MFN1032 on enterocytes can be modulated by serotonin, substance P and epinephrine. Arch Microbiol. 2015; 197(8): 983–990. PubMed Abstract \| Publisher Full Text\n\nBlair JM, Richmond GE, Piddock LJ: Multidrug efflux pumps in Gram-negative bacteria and their role in antibiotic resistance. Future Microbiol. 2014; 9(10): 1165–1177. PubMed Abstract \| Publisher Full Text\n\nBlanco P, Hernando-Amado S, Reales-Calderon JA, et al.: Bacterial multidrug efflux pumps: much more than antibiotic resistance determinants. Microorganisms. 2016; 4(1): 14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBreidenstein EB, de la Fuente-Núñez C, Hancock RE: Pseudomonas aeruginosa: all roads lead to resistance. Trends Microbiol. 2011; 19(8): 419–426. PubMed Abstract \| Publisher Full Text\n\nEvans EA, Kawli T, Tan MW: Pseudomonas aeruginosa suppresses host immunity by activating the DAF-2 insulin-like signaling pathway in Caenorhabditis elegans. PLoS Pathog. 2008; 4(10): e1000175. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFernández M, Porcel M, de la Torre J, et al.: Analysis of the pathogenic potential of nosocomial Pseudomonas putida strains. Front Microbiol. 2015; 6: 871. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGonzalez MR, Ducret V, Leoni S, et al.: Pseudomonas aeruginosa zinc homeostasis: Key issues for an opportunistic pathogen. Biochim Biophys Acta Gene Regul Mech. 2019; 1862(7): 722–733. PubMed Abstract \| Publisher Full Text\n\nHancock RE, Speert DP: Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and impact on treatment. Drug Resist Updat. 2000; 3(4): 247–255. PubMed Abstract \| Publisher Full Text\n\nKawai Y: Purification and characterization of pertucin produced by Pseudomonas pertucinogena. Antimicrob Agents Chemother. 1974; 6(3): 347–359. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLalucat J, Bennasar A, Bosch R, et al.: Biology of Pseudomonas stutzeri. Microbiol Mol Biol Rev. 2006; 70(2): 510–547. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi XZ, Plésiat P, Nikaido H: The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria. Clin Microbiol Rev. 2015; 28(2): 337–418. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSadikot RT, Blackwell TS, Christman JW, et al.: Pathogen–host interactions in Pseudomonas aeruginosa pneumonia. Am J Respir Crit Care Med. 2005; 171(11): 1209–1223. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSilby MW, Winstanley C, Godfrey SA, et al.: Pseudomonas genomes: diverse and adaptable. FEMS Microbiol Rev. 2011; 35(4): 652–680. PubMed Abstract \| Publisher Full Text\n\nSoto SM: Role of efflux pumps in the antibiotic resistance of bacteria embedded in a biofilm. Virulence. 2013; 4(3): 223–229. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWebber M, Piddock L: The importance of efflux pumps in bacterial antibiotic resistance. J Antimicrob Chemother. 2003; 51(1): 9–11. PubMed Abstract \| Publisher Full Text\n\nXin XF, Kvitko B, He SY: Pseudomonas syringae: what it takes to be a pathogen. Nat Rev Microbiol. 2018; 16(5): 316. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "68814", "date": "19 Aug 2020", "name": "Asad U. Khan", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nShort communication focuses on antibiotic resistance caused by efflux pump overexpression and the use of cryo electron microscopy for protein protein or protein ligand interaction. It also describes the limitations of x ray crystallography. The author also extends the cryo EM use in interparticle motions of the ribosome in different tRNA bound states, its visualization in various conformations upon the introduction of ribosomal-specific substrates and several bacterial membrane proteins to near atomic resolution. Author successfully explain the utility of structural dynamics in drug designing by identifying important residues through solved structures. However, references are missing from the introduction and targeting efflux pumps to mitigate antibiotic resistance section. May be accepted after minor revision.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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 source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "74062", "date": "16 Nov 2020", "name": "Paula Blanco", "expertise": [ "Reviewer Expertise Antimicrobial resistance evolution" ], "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 brief report deals with the comparison of five Pseudomonas species' genomes in order to get information about the different antibiotic resistance genes and mechanisms. However, the findings described here lack novelty and interest, since the mechanisms of resistance and antimicrobial resistance genes have been widely described in the different Pseudomonas species along the literature. Another point to consider is the number of genomes used for the work. Five complete genomes as a representation of the Pseudomonas pathogens group are not enough for drawing conclusions. It would be much more interesting to take a larger sample of genomes (since they are available) including more than a strain per Pseudomonas species, as well as environmental and clinical strains. A more extensive and careful use of literature should be used along the manuscript, since there are some sentences that does not include a reference. For instance \"The most common resistance mechanism of Pseudomonas pathogens is the antibiotic efflux pump mechanism\". Also, one of the references for the sentence \"P. aeruginosa is of great medical importance due to its exhibition of multidrug resistance and its association with serious illnesses\" mention a work performed in the nematode C. elegans. It would be right to include more references related to the clinics. Other aspects of this brief report should need clarification, for instance, what is RG in Table 1? What is the difference between perfect and strict cutoff in Fig1A? Figure 2B also needs clarification, since it is difficult for the reader to make an interpretation and get any conclusion. The author claim in the conclusion that the \"findings of this brief report could be useful in understanding the chemotherapeutics against antibiotic resistance strains of Pseudomonas pathogens\". However, it is not discussed how actually these data could be used or applied. The author should also mention other similar in silico studies already published to compare his results and give consistency to the methodology employed here.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-903
https://f1000research.com/articles/9-901/v1	04 Aug 20	{ "type": "Study Protocol", "title": "Lifestyle and reproductive health: the aetiology of ovarian cancer in Pakistan", "authors": [ "Qurratulann Alvi", "Gul Muhammad Baloch", "Karuthan Chinna", "Ali Dabbagh", "Qurratulann Alvi", "Karuthan Chinna", "Ali Dabbagh" ], "abstract": "Ovarian cancer is a fatal gynaecological cancer and eighth most common cancer in women globally. Lifestyle, reproductive and sociodemographic factors are among the influential parameters that may significantly affect the risk of ovarian cancer and its mortality rate. However, the epidemiological investigations have shown that the risk of ovarian cancers associated with these factors is different in varied geographical distributions. Lifestyle and reproductive factors have not been investigated thoroughly across a wide cultural diversity. The objective of this study is to investigate the association of these factors with ovarian cancer in Pakistan. This investigation will focus on the lifestyle effects of fat intake, intake of tea, habitual exercise, use of talc, personal hygiene, habit of holding urine for long time, obesity on ovarian cancer among Pakistani women. Reproductive variables will include age at menarche, natural menopausal age, parity, nulliparity (miscarriages, abortion, stillbirths), infertility, fertility treatment, tubal ligation, oral contraceptive use, and family history of breast or ovarian cancer. Sociodemographic variables will include effect of age, income, education, and geographical location. A case-control study will be conducted in the major cancer hospitals of Pakistan and the patients will also be interviewed. The controls will be recruited outside the hospital. For controls the same age limit and residency requirements will be applied. The information gained from this research will be an important contribution to develop programs for health promotion, with a focus on ovarian cancer prevention and women’s health. The findings could be used for health policies and planning to prevent ovarian cancer. The research will pave the way for a public policy and interventions to reduce the burden of ovarian cancer in Pakistan.", "keywords": [ "Ovarian Cancer", "Reproductive Health", "Lifestyle", "Miscarriage", "Pakistan" ], "content": "Introduction\n\nOvarian cancer is one of the most frequently fatal gynaecologic cancers (Jayson et al., 2014; Tworoger & Huang, 2016). According to the American Institute for Cancer Research report, in 2018 ovarian cancer globally accounted for 3.6% of all forms of cancers and the eighth leading cause of death among women globally (Merritt et al., 2018). Only 46% of ovarian cancer patients survive beyond 5 years (Kathawala et al., 2018). The high mortality associated with ovarian cancer is due to late diagnosis and resistance to treatment, (Carollo et al., 2019; Nunes et al., 2019).\n\nAccording to a 2018 report by World Cancer Research Fund, the diagnosed cases of ovarian cancer are 295,414. The estimated age-standardized incidence and mortality rates of ovarian cancer in 2018 were 6.6 and 3.9, respectively (Bray et al., 2018) and this number is expected to reach at 434,184 by 2040.\n\nThe incidence, prevalence, and mortality of ovarian cancer varies across geographical locations and countries (Coburn et al., 2017). Globally, the highest incidence and mortality for ovarian cancer was reported in Serbia (16.6 and 6.8, respectively) and lowest in Gambia (0.6 and 0.43, respectively); in Europe itself, figures were highest in Serbia and lowest in Ireland (11.4 and 6.4, respectively); in Asia, rates are highest in Brunei (16 and 6.2, respectively) and lowest in Yemen (2.6 & 2.1, respectively) (https://gco.iarc.fr/today/online-analysis-table).\n\nThe epidemiological diversity could be linked to different risk factors for ovarian cancer (Hunn & Rodriguez, 2012). Poole et al. (2013) called for identifying modifiable risk factors. Jammal et al. (2017) believe that ovarian cancer prevention can be accomplished with clinical approaches.\n\nThe aetiology of ovarian cancer cannot be defined by single mechanism (Terry & Missmer, 2017). Hence understanding of risk factors is important (Webb & Jordan, 2017). Clinical evidence shows that by eliminating and decreasing the risk factors, the ovarian cancer cases can be prevented (Bray et al., 2018). Lifestyle modifications can minimize cancer burden (Song & Giovannucci, 2016).\n\nAli (2018) noted that knowledge on ovarian cancer might increase survival rate. This can help women at risk of ovarian cancer to take special precautions (Li et al., 2015; Torre et al., 2018); thus, help reduce ovarian cancer risk (Momenimovahed et al., 2019).\n\nSchildkraut et al. (2019) reported obesity and family history of breast cancer to be major risk factors for ovarian cancer and Abbott et al. (2016) reported inadequacy of physical activity to be a risk factor. Gabriel et al. (2019) found the use of talc in genital areas to be a risk factor for ovarian cancer in England. In a Canadian study, Koushik et al. (2017) showed strong inverse association between parity and ovarian cancer. Harris et al. (2017) reported oral contraceptive and family history as the main risk factors in an American population. Lee et al. (2013) showed that consumption of green tea increases ovarian cancer risk in a Chinese population. Other risk factors include infertility (Rasmussen et al., 2017), miscarriages (Moorman et al., 2016) and age at menopause.\n\nDoherty et al. (2017) suggests role of ethnically differing populations. Endpoints must represent the disease agent involved and rely on the quality of life (Wilson et al., 2017). Responsive and timely health care facilities that use relational communication, could enhance women's health experience with ovarian cancer (Jelicic et al., 2019).\n\nThis study explores lifestyle (use of talc, obesity, pattern of weight change), reproductive health (miscarriages, age at menarche, menopausal age, parity, nulliparity) and socio-demographic factors (age, income, and geographical location) associated with ovarian cancer in Pakistan.\n\n\nMethods\n\nThe research is now at an advanced stage after a development of a literature review, methodology and preparation of a validated questionnaire. The study will be conducted from July 2020 to December 2020. Data collection will start in July 2020.\n\nThis will be a case-control study. The cases will be ovarian cancer patients registered at cancer hospitals in Pakistan. Hospitals are selected based on receiving approval from the hospital administrations. The controls will be recruited from the general population, using random digit dialling of individuals in the vicinity of the selected hospitals, since this was what the participating hospitals wanted. Cases and controls will not be matched. In the analysis, the outcome variables will be corrected for all the variables. We do not have the ability to mitigate many sources of bias as we must comply with the hospital’s’ requirements.\n\nA validated questionnaire (validated by a panel of experts, each with doctorates and many years’ experience in biostatistics, public health and biomedicine) will be used to elicit information on following parameters.\n\n1. Socio demographic characteristics\n\n2. Patient clinical data\n\n3. Diagnostic data\n\n4. Lifestyle factors\n\n5. Reproductive health\n\nPatients’ clinical and diagnostic data will be obtained from the respective hospital registries. Written informed consent will be obtained from all participants with the voluntary decision to participate in the study. Interviews will be conducted with questions relevant to the parameters mentioned above.\n\nPower and Sample Size software was used to calculate sample size. The level of significance and power were set as 0.05 and 80%, respectively. Based on results, the minimum required sample size is 387 for cases and 387 for controls. The figures are rounded up to 400 per group.\n\nInclusion criteria for cases\n\n• Age 30 to 65\n\n• Confirmed diagnosis of ovarian cancer by hospital or health care facility.\n\nExclusion criteria for cases\n\n• Cognitively impaired\n\n• Diagnosed with any other form of cancer\n\nThe inclusion criteria for control will be, age 30 to 65, healthy and cognitively not impaired; and exclusion criteria will be comorbidities, such as diabetes or cardiovascular disease.\n\nData will be analysed using SPSS version 25. Qualitative variables will be described as frequencies and percentages, while quantitative variables will be described as means and standard deviations if the variable is normally distributed and as median and interquartile ranges, otherwise. Univariate and multivariate binary logistic regression analyses will be used in testing association between the predictor variables and ovarian cancer. In the univariate analysis, the predictor variables will be tested on at time. The variables that are significant at 0.25 level will be included in the multivariate analysis. Finally, stepwise analysis will be used to determine the significant predictors of ovarian cancer.\n\nEthical approval for this study will be obtained from the participating hospitals/authorities in Pakistan. Respondents will be briefed, consent obtained and the confidentiality and participants’ right to stop at any point of the study will be assured.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nAbbott SE, Bandera EV, Qin B, et al.: Recreational physical activity and ovarian cancer risk in African American women. Cancer Med. 2016; 5(6): 1319–1327. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAli AT: Towards Prevention of Ovarian Cancer. Curr Cancer Drug Targets. Bentham Science Publishers. 2018; 18(6): 522–537. PubMed Abstract \| Publisher Full Text\n\nBray F, Ferlay J, Soerjomataram I, et al.: Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018; 68(6): 394–424. PubMed Abstract \| Publisher Full Text\n\nCarollo E, Paris B, Samuel P, et al.: Detecting ovarian cancer using extracellular vesicles: progress and possibilities. Biochem Soc Trans. 2019; 47(1): 295–304. PubMed Abstract \| Publisher Full Text\n\nCoburn SB, Bray F, Sherman ME, et al.: International patterns and trends in ovarian cancer incidence, overall and by histologic subtype. Int J Cancer. Wiley Online Library, 2017; 140(11): 2451– 2460. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEpidemiology Working Group Steering Committee, Ovarian Cancer Association Consortium Members of the EWG SC, Doherty JA, et al.: Current Gaps in Ovarian Cancer Epidemiology: The Need for New Population-Based Research. J Natl Cancer Inst. Oxford University Press, 2017; 109(10): djx144.PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGabriel IM, Vitonis AF, Welch WR, et al.: Douching, Talc Use, and Risk for Ovarian Cancer and Conditions Related to Genital Tract Inflammation. Cancer Epidemiol Biomarkers Prev. 2019; 28(11): 1835–1844. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarris HR, Titus LJ, Cramer DW, et al.: Long and irregular menstrual cycles, polycystic ovary syndrome, and ovarian cancer risk in a population-based case-control study. Int J Cancer. 2017; 140(2): 285–291. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHunn J, Rodriguez GC: Ovarian cancer: etiology, risk factors, and epidemiology. Clin Obstet Gyneco. LWW 2012; 55(1): 3–23. PubMed Abstract \| Publisher Full Text\n\nJammal MP, de Lima CA, Nomelini RS, et al.: Is Ovarian Cancer Prevention Currently Still a recommendation of Our Grandparents? Rev Bras Ginecol Obstet. ThiemeRevinterPublicaç\\~oesLtda. 2017; 39(12): 676–685. PubMed Abstract \| Publisher Full Text\n\nJayson GC, Kohn EC, Kitchener HC, et al.: Ovarian cancer. Lancet. 2014; 384(9951): 1376–1388. PubMed Abstract \| Publisher Full Text\n\nJelicic L, Brooker J, Shand L, et al.: Experiences and health care preferences of women with ovarian cancer during the diagnosis phase. Psychooncology. Wiley Online Library, 2019; 28(2): 379–385. PubMed Abstract \| Publisher Full Text\n\nKathawala RJ, Kudelka A, Rigas B: The Chemoprevention of Ovarian Cancer: the Need and the Options. Curr Pharmacol Rep. Springer, 2018; 4(3): 250–260. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoushik A, Grundy A, Abrahamowicz M, et al.: Hormonal and reproductive factors and the risk of ovarian cancer. Cancer Causes Control. 2017; 28(5): 393–403. PubMed Abstract \| Publisher Full Text\n\nLee AH, Su D, Pasalich M, et al.: Tea consumption reduces ovarian cancer risk. Cancer Epidemiol. 2013; 37(1): 54–59. PubMed Abstract \| Publisher Full Text\n\nLi K, Hüsing A, Fortner RT, et al.: An epidemiologic risk prediction model for ovarian cancer in Europe: the EPIC study. Br J Cancer. Nature Publishing Group, 2015; 112(7): 1257–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMerritt MA, Rice MS, Barnard ME, et al.: Pre-diagnosis and post-diagnosis use of common analgesics and ovarian cancer prognosis (NHS/NHSII): a cohort study. Lancet Oncol. Elsevier, 2018; 19(8): 1107–1116. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMomenimovahed Z, Tiznobaik A, Taheri S, et al.: Ovarian cancer in the world: epidemiology and risk factors. Int J Womens Health. Dove Press, 2019; 11: 287–299. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoorman PG, Alberg AJ, Bandera EV, et al.: Reproductive factors and ovarian cancer risk in African-American women. Ann Epidemiol. 2016; 26(9): 654–662. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNunes AS da A de, Serpa J, Félix A, et al.: Cysteine, a facilitator of hypoxia adaptation and a promoter of drug-resistance: a new route to better diagnose and treat ovarian cancer patients. 2019. Reference Source\n\nPoole EM, Merritt MA, Jordan J, et al.: Hormonal and reproductive risk factors for epithelial ovarian cancer by tumor aggressiveness. Cancer Epidemiol Biomarkers Prev. AACR, 2013; 22(3): 429–437. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRasmussen ELK, Hannibal CG, Dehlendorff C, et al.: Parity, infertility, oral contraceptives, and hormone replacement therapy and the risk of ovarian serous borderline tumors: A nationwide case-control study. Gynecol Oncol. 2017; 144(3): 571–576. PubMed Abstract \| Publisher Full Text\n\nSchildkraut JM, Peres LC, Bethea TN, et al.: Ovarian Cancer in Women of African Ancestry (OCWAA) consortium: a resource of harmonized data from eight epidemiologic studies of African American and white women. Cancer Causes Control. 2019; 30(9): 967–978. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSong M, Giovannucci E: Preventable Incidence and Mortality of Carcinoma Associated With Lifestyle Factors Among White Adults in the United States. JAMA Oncol. American Medical Association, 2016; 2(9): 1154–1161. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTerry KL, Missmer SA: Epidemiology of ovarian and endometrial cancers. In: Pathology and Epidemiology of Cancer. Springer, 2017; 233–246. Publisher Full Text\n\nTorre LA, Trabert B, DeSantis CE, et al.: Ovarian cancer statistics, 2018. CA Cancer J Clin. Wiley Online Library, 2018; 68(4): 284–296. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTworoger SS, Huang T: Obesity and ovarian cancer. In: Obesity and Cancersity and Cancer. Springer, Cham. 2016; 155–176. PubMed Abstract \| Publisher Full Text\n\nWebb PM, Jordan SJ: Epidemiology of epithelial ovarian cancer. Best Pract Res Clin Obstet Gynaecol. Elsevier, 2017; 41: 3–14. PubMed Abstract \| Publisher Full Text\n\nWilson MK, Pujade-Lauraine E, Aoki D, et al.: Fifth Ovarian Cancer Consensus Conference of the Gynecologic Cancer InterGroup: recurrent disease. Ann Oncol. Oxford University Press, 2017; 28(4): 727–732. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "68702", "date": "07 Aug 2020", "name": "Ramesh Kumar", "expertise": [ "Reviewer Expertise 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\nThis will be an important study proposal and will be helpful for future research evidence. Ovarian cancer is a big problem in Pakistan and researchers are strongly needed to work in this area. Moreover, the author has proposed a case-control study that would explore multiple factors responsible to develop this problem in Pakistan.\n\nThis is a study protocol developed by the author and the author will collect the data and publish it later on\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": [] }, { "id": "68704", "date": "10 Aug 2020", "name": "Sadiq Bhanbhro", "expertise": [ "Reviewer Expertise Public health", "social sciences", "research methods." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-designed study protocol aimed to investigate the relationships between lifestyle factors, reproductive health, and risk of ovarian cancer in Pakistan. The aims, objectives and study design including sample size and data collection methods are appropriate and sufficiently described. The authors have cited the current and relevant literature. However, considering the sensitive nature of the topic under study, I would suggest the investigators need to provide more details on the recruitment of the potential study participants and the ethical considerations. For instance, the authors’ need to identify and name the relevant academic ethics review committee that will approve the project.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/9-901
https://f1000research.com/articles/9-376/v1	19 May 20	{ "type": "Software Tool Article", "title": "Jupyter notebook-based tools for building structured datasets from the Sequence Read Archive", "authors": [ "Matthew N. Bernstein", "Ariella Gladstein", "Khun Zaw Latt", "Emily Clough", "Ben Busby", "Allissa Dillman", "Ariella Gladstein", "Khun Zaw Latt", "Emily Clough", "Ben Busby", "Allissa Dillman" ], "abstract": "The Sequence Read Archive (SRA) is a large public repository that stores raw next-generation sequencing data from thousands of diverse scientific investigations. Despite its promise, reuse and re-analysis of SRA data has been challenged by the heterogeneity and poor quality of the metadata that describe its biological samples. Recently, the MetaSRA project standardized these metadata by annotating each sample with terms from biomedical ontologies. In this work, we present a pair of Jupyter notebook-based tools that utilize the MetaSRA for building structured datasets from the SRA in order to facilitate secondary analyses of the SRA’s human RNA-seq data. The first tool, called the Case-Control Finder, finds suitable case and control samples for a given disease or condition where the cases and controls are matched by tissue or cell type. The second tool, called the Series Finder, finds ordered sets of samples for the purpose of addressing biological questions pertaining to changes over a numerical property such as time. These tools were the result of a three-day-long NCBI Codeathon in March 2019 held at the University of North Carolina at Chapel Hill.", "keywords": [ "Hackathon", "RNA-seq", "Sequence Read Archive", "MetaSRA", "Metadata", "Ontology", "Jupyter" ], "content": "Introduction\n\nThe Sequence Read Archive (SRA; Leinonen et al., 2011) is a large public repository that stores next-generation sequencing data from thousands of diverse scientific investigations. Despite its promise, reuse and re-analysis of SRA data has been challenged by the heterogeneity and poor quality of the metadata that describe its biological samples (Gonçalves & Musen, 2019). Recently, the MetaSRA project (Bernstein et al., 2017) standardized these metadata by annotating each sample with terms from biomedical ontologies including Cell Ontology (Bard et al., 2005), Uberon (Mungall et al., 2012), Disease Ontology (Schriml et al., 2019), Cellosaurus (Bairoch, 2018), and the Experimental Factors Ontology (Malone et al., 2010). The MetaSRA also features an interface (http://metasra.biostat.wisc.edu) for querying human RNA-seq samples using these ontology term annotations. However, the MetaSRA web interface is not capable of producing structured datasets such as those that match case samples associated with a target condition or disease with healthy control samples. Similarly, the MetaSRA is also not capable of searching for samples associated with a particular condition and/or tissue-type that are ordered according to a numeric property (e.g., age).\n\nConstruction of such datasets is non-trivial and requires further processing of the results provided by the MetaSRA website. For example, finding case and control samples for a given disease likely requires matching case samples to control samples according to their tissue or cell type. Furthermore, given these search results, users may wish to further filter samples according to whether they are poorly annotated (i.e., are missing cell type or tissue information), whether they are derived from a cell line, or whether they were experimentally treated. Moreover, given these results, the user may wish to explore other ontology terms associated with the search results within either the case or control samples to check for any variables that may confound downstream analyses. Finding longitudinal or time-series data presents similar challenges. To the best of our knowledge, no existing tool addresses these tasks.\n\nTo address these two tasks, we produced two Jupyter notebook-based tools. The first tool, called the Case-Control Finder, searches the SRA via the MetaSRA terms to produce matched-case and control samples for a given disease or condition where the cases and controls are matched by tissue and cell type. The second tool, called the Series Finder, finds ordered sets of samples for the purpose of answering biological questions pertaining to changes over a numerical property (e.g., time). More specifically, the Series Finder produces ordered sets of samples, where the order is determined based on a temporal property in the metadata such as age, as standardized by the MetaSRA’s real-valued properties. These tools promise to facilitate the construction of suitable public datasets for secondary analyses.\n\n\nMethods\n\nThe tools presented in this work were written in Python (v3.6) and make use of Python packages pandas (McKinney, 2011), Matplotlib (Hunter, 2007), and seaborn (https://seaborn.pydata.org). These notebooks are available ready-to-run in a Docker container.\n\nThe Case-Control Finder implements the following steps to produce a dataset of matched-case control samples for a given disease (Figure 1A):\n\nAn overview of the backend processing functions called from the Jupyter notebooks.\n\n1. Generate candidate case and control samples. Generate the set of candidate case samples by querying for all samples associated with a user-specified condition or disease using the MetaSRA-mapped ontology terms. Also, find all candidate control samples that are not associated with the target condition/disease.\n\n2. Filter poorly annotated samples. Filter samples based on a metadata completeness threshold, which requires that all samples be associated with either a tissue term or a cell type term. The tissue/cell type information is required for downstream matching of case samples to control samples.\n\n3. Apply user-specified filters. Further filter samples according to user-specified filtering parameters. The user can filter out cell line samples, treated samples, and in vitro differentiated samples. The user can also remove all diseased samples from the candidate control samples for the purpose of generating a healthy control-set.\n\n4. Match by tissue and cell type. The candidate case samples are then matched with the candidate control samples by their tissue and cell type terms. Specifically, given that each sample can be associated with multiple ontology terms in the MetaSRA, a set of case samples is matched with a set of control samples when both sets of samples are labelled with the same set of tissue and cell type terms. For example, a set of case samples annotated with the set of terms “liver” and “epithelial cell” will be matched only to control samples also labeled strictly with these terms (Figure 2A). This ensures that case samples are matched with maximally similar control samples and mitigates matching samples from different tissue-types. For example, a set of case samples labelled with both the terms “liver” and “epithelial cell” will not be matched with a set of samples labelled only as “epithelial cell,” as there is no guarantee that the latter set of samples originate in the liver.\n\nResults from running the Case-Control Finder for the query “liver cancer.” (A) The Case-Control Finder displays the number of case/control studies (left) and case/control samples (right) matched by each tissue and cell type. (B) The user can select either the case samples or control samples for a given tissue or cell type and display the most common ontology terms associated with those selected samples. Displayed here are the most common terms associated with the case samples labeled as “liver.” (C) The notebook also displays four pie charts for viewing the fraction of samples belonging to a cell line (top left), each sex (top right), each developmental stage (bottom left), and whether they were given an experimental treatment (bottom right).\n\nOnce the dataset is constructed, the notebook enables the user to explore the samples for other MetaSRA mapped ontology terms within the data (Figure 2B and C). By presenting other common ontology terms in the data, the user may be able to identify variables that either confound analysis.\n\nThe Series Finder finds RNA-seq data samples that are associated with a numerical property (e.g., age or time point) for a given tissue or cell type. To do so, the Series Finder utilizes the real-value property annotations provided by the MetaSRA where each real-value property in the MetaSRA is structured as a tuple consisting of a property name (e.g., age), numerical value, and unit (e.g., year).\n\nTo perform a query, the user provides an ontology term, such as a tissue or cell type, as well as a property name and unit. The Series Finder then finds all samples that are associated with the target ontology term and real-value property. The user can also provide a set of blacklist terms that can be used to filter the samples. Given a list of blacklist terms, the Series Finder will remove all samples annotated with any blacklist term. The Series Finder will then return all remaining samples ordered by their associated numerical values (Figure 1B).\n\n\nResults and use cases\n\nWe used the Case-Control Finder to query for samples of liver cancer RNA-seq samples matched with healthy control samples. This query resulted in six sets of samples representing different tissues or cell types including epithelial cells, hepatocytes, stem cells, and liver tissue (Figure 2A). The Case-Control Finder identified common terms associated with the case “liver cancer” samples (Figure 2B), and categorized these samples by cell line status, sex, developmental stage, and treatment status (Figure 2C).\n\nWe used the Series Finder to find all brain samples in the SRA ordered by the age of the sample donor. This query resulted in samples spanning many ages (Figure 3A). This dataset could prove useful for exploring gene expression-based signatures of aging. The Series Finder also identified common terms at each age (Figure 3B) and for each age’s sample-set, categorized those samples by cell line status, sex, developmental stage, and treatment status (Figure 3C).\n\nResults from running the Series Finder for the query “brain” sorted by “age,” where unit is specified as “year.” (A) The Series Finder displays the number of samples sorted by age. (B) The user can select samples associated with a given time point for further exploration. Here the samples annotated as “year = 63” are selected. The notebook then displays four pie charts for viewing the fraction of samples belonging to a cell line (top left), each sex (top right), each developmental stage (bottom left), and whether they were given an experimental treatment (bottom right). (C) Given the selected samples from (B), the notebook displays the most frequent terms associated with those selected samples. Displayed here are the most frequent terms associated with the case samples labeled as “liver.”\n\n\nConclusion and future work\n\nWe implemented two Jupyter notebooks for performing hypothesis-driven queries of public RNA-seq samples in the SRA. These tools are built upon the standardized metadata provided by the MetaSRA project and enable querying of the metadata beyond what is natively possible via the MetaSRA website interface. Future work will entail either integrating these tools into a standard web-interface, such as the interface of the MetaSRA website, or by implementing a stand-alone web application for these tools using a platform such as R Shiny.\n\n\nData availability\n\nThe figures and datasets produced in the analyses can be found on GitHub: https://github.com/mbernste/hypothesis-driven-SRA-queries/tree/master/results\n\n\nSoftware availability\n\nAll code is maintained on GitHub: https://github.com/mbernste/hypothesis-driven-SRA-queries\n\nArchived code as at time of publication: https://doi.org/10.5281/zenodo.3807512 (Bernstein, 2020)\n\nLicense: CC0", "appendix": "Acknowledgements\n\nWe would like to thank Carl Leubsdorf and Brad Plecs for technical support using Google Cloud Platform servers, and J. Rodney Brister and Barton Trawick for administrative support.\n\n\nReferences\n\nBairoch A: The Cellosaurus, a Cell-Line Knowledge Resource. J Biomol Tech. 2018; 29(2): 25–38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBard J, Rhee SY, Ashburner M: An ontology for cell types. Genome Biol. 2005; 6(2): R21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBernstein M: mbernste/hypothesis-driven-SRA-queries: First release (Version v1.0.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3807512\n\nBernstein MN, Doan A, Dewey CN: MetaSRA: normalized human sample-specific metadata for the Sequence Read Archive. Bioinformatics. 2017; 33(18): 2914–2923. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGonçalves RS, Musen MA: The variable quality of metadata about biological samples used in biomedical experiments. Scientific Data. 2019; 6: 190021. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHunter JD: Matplotlib: A 2D graphics environment. Comput Sci Eng. 2007; 9(3): 90–95. Publisher Full Text\n\nLeinonen R, Sugawara H, Shumway M, et al.: The Sequence Read Archive. Nucleic Acids Res. 2011; 39(Database issue): D19–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMalone J, Holloway E, Adamusiak T, et al.: Modeling sample variables with an Experimental Factor Ontology. Bioinformatics. 2010; 26(8): 1112–1118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcKinney W: pandas: a foundational Python library for data analysis and statistics. Python for High Performance and Scientific Computing. 2011; 14. Reference Source\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\nSchriml LM, Mitraka E, Munro J, et al.: Human Disease Ontology 2018 update: classification, content and workflow expansion. Nucleic Acids Res. 2019; 47(D1): D955–D962. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "63613", "date": "01 Jun 2020", "name": "Zichen Wang", "expertise": [ "Reviewer Expertise Bioinformatics", "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\nBernstein et al. provides two Jupyter notebook-based tools to facilitate re-analysis of human RNA-seq data deposited to SRA. The tools were built on top of annotated metadata of RNA-seq samples from the MetaRNA, and provided some visualizations of the summary statistics of the query results.\nI have the following suggestions and comments:\nThe authors should indicate how to access the Jupyter notebooks in the abstract.\n\nIt would require less overhead for users if the authors make their Jupyter notebook tools available to execute on Binder or Google Colab.\n\nSince MetaSRA mapped RNA-seq samples to biomedical ontologies, it would be useful to have the Jupyter tools also enable query using ontology terms in addition to free texts. For instance, a researcher may want to focus on samples from non-small cell lung carcinoma (DOID:3908) rather than any types of lung cancers.\n\nCurrently, both notebooks load the metadata of the SRA samples from a preprocessed file in the Git repository. It would be useful to make it interoperable with MetaSRA through API to be able to query against the most updated version of SRA, which may include many more samples. As the volume of public RNA-seq data are drastically increasing.\n\nPlease provide available options for the structured query, including \"target_property\" and \"UNIT\", in the \"Series Finder\" notebook.\n\nPlease provide assessment of the precision and recall of the tools in terms of retrieving the correct samples given queries.\n\nCan the authors please comment on the applicability of the tools on bulk vs. single-cell samples?\n\nPlease add discussion about how to perform secondary analysis on the SRA samples after obtaining the structured data from the Jupyter notebooks.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "5762", "date": "04 Aug 2020", "name": "Matthew Bernstein", "role": "Author Response", "response": "We greatly appreciate the reviewer's valuable feedback. Please find our responses to each point below:1. Within the abstract we now point the reader to the tools’ Github repository, which describes how the tools can be executed either locally or in the cloud via Google Colab.2. We have set up Google Colab notebooks to run these tools in the cloud. Links to the notebooks are found within the README in the Github repository.3. We thank you for this suggestion. We have updated the tools to now accept both ontology term names (i.e. free text) as well as ontology term ID’s.4. We agree that using the MetaSRA’s API would be a great idea; however, the API restricts queries that return too many results. Specifically, for queries that return too many results, the API returns an error message that the search results are too large. This severely restricts our ability to use the API for these tools. We note that the MetaSRA is released in discrete chunks and does not track every ongoing change to the SRA; thus, whenever the MetaSRA version changes, we will update the static version of the MetaSRA packaged with these tools. We have added text to this manuscript detailing our commitment to performing these updates. Lastly, we added text to the README that makes it more explicit to the user which version of the MetaSRA these tools are utilizing.5. Within the instructions (within Section 1 of the Series Finder), we now provide the user example properties (such as “passage number” and “time”) as well as example units (such as “hour” and “day”). We also point the user to the Units Ontology for a full set of available units that are utilized by the underlying MetaSRA annotations.6. We note that the accuracy of the results is dependent on the accuracy of the MetaSRA annotations, which have been thoroughly evaluated in the original MetaSRA publication by Bernstein et al. (2017). Therefore, we added text to the “Conclusion and future work” section that points readers to this analysis. We have also added text to this section that clarifies that these tools are for selecting an initial candidate set of samples from the SRA; however, given that the annotations are not error-free, we encourage the user to further validate the datasets returned by these tools before performing downstream analysis.7. The SRA stores sequencing data for both bulk and single-cell data; however, this information is not encoded in the metadata in a standardized way nor is it captured by the MetaSRA. Therefore, one limitation of the tools presented in this work is that they may return datasets that comprise both bulk and single-cell samples. We describe this limitation in the Conclusion section and again encourage users to validate the results returned by these tools before performing downstream analyses.8. In the Conclusion section, we now point the reader to databases of pre-processed SRA data including recount2, ARCHS4, and refine.bio. From these resources, users can download pre-processed expression data for the samples returned by the tools presented in this work." } ] }, { "id": "63614", "date": "05 Jun 2020", "name": "Shannon Ellis", "expertise": [ "Reviewer Expertise genetics", "bioinformatics", "data science 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\nThis paper describes the development of two Jupyter notebook-based tools (Case-Control Finder and Series Finder) for improving the ease with which researchers can identify cases within the SRA for further study.\nWhile the paper does a nice job describing what the tool is and how it can be helpful and the code & examples provided/explained the paper function as expected (is reproducible), there are a few limitations in its implementation that will limit its utility with researchers:\n\nThe fact that this tool requires a static version of the SRA metadata to be loaded in limits its ability to be updated and requires the authors to manually download the metadata - access by API to SRA would improve this process.\n\nWhile the provided examples work well, there are limitations to unfamiliar users and failures in cases that seem on reading the paper like they should work. - For example: in series finder if I change `term` to \"heart\" (instead of \"brain\"), almost all subsequent cells fail. - In case-control finder, if I change `condition` to \"brain cancer\", all but one samples returned are controls (which does not align with what is in the SRA?) and visualization formatting becomes difficult. - By clarifying what user options are (or examples) for each place where user is free to play with the input, this could be avoided. Similarly, functions lack documentation and examples here or checks on input within the functions, so diving into the code becomes critical for use, which will limit users. Adding documentation and checks for user input could assist in this overall.\n\nMinor issues:\nI was able to download locally using the \"not recommended\" approach; however, docker asked for a password using suggested approach in README (I didn't investigate further).\n\nIn the paper & notebooks, tool would be improved by focusing on readability of visualizations. For example, flipping the bar charts in figure 2A by 90 degrees (and accompanying in the notebook), the labels would be more readable. And, by considering the colors in figure C, such that \"orange\" is not used in all three pie charts (when they do not represent the same categories) would be helpful. Having the number of samples summarized by the pie charts would also be helpful.\n\nThe sentence in Introduction starting with \"More specifically, the Series Finder produces...\" is unclear. Specifically, on reading, I'm not sure what a temporal property would be in the metadata (other than the listed age). As a reader, this limits my understanding of 1 of the two notebooks provided and my ability to use the tool.\n\nI may be missing it, but it seems like cases and controls would benefit most from being able to also be matched on age and sex to truly make them useful for further analysis. It does not seem this functionality exists, or I'm missing it.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "5763", "date": "04 Aug 2020", "name": "Matthew Bernstein", "role": "Author Response", "response": "We greatly appreciate the reviewer's valuable suggestions and feedback. Please see our responses below:1. We agree that using the MetaSRA’s API would be a great idea; however, the API restricts queries that return too many results. Specifically, for queries that return too many results, the API returns an error message that the search results are too large. This severely restricts our ability to use the API for these tools. We note that the MetaSRA is released in discrete chunks and does not track every ongoing change to the SRA; thus, whenever the MetaSRA version changes, we will update the static version of the MetaSRA packaged with these tools. We have added text to this manuscript detailing our commitment to performing these updates. Lastly, we added text to the README that makes it more explicit to the user which version of the MetaSRA these tools are utilizing.2. - We tested the query “heart” and it now should return results. We also provide more thorough input validation for cases in which the query does not return results.- We have updated the code so that the tools retrieves sample that are annotated as an ancestral term to the query term (e.g. a sample labelled as “brain glioma” should be retrieved when the user inputs the query “brain cancer”). Now the query “brain cancer” will retrieve many more samples than before. We do note a few issues with the particular query “brain cancer” (which maps to term DOID:1319 in the Disease Ontology). Specifically, we found that the MetaSRA failed to label many samples as “brain cancer” due to the fact that many of the subterms (e.g. “brain glioma”) are missing important synonyms that would have led the MetaSRA to pick them up. For example, the term “brain glioma” (DOID:0060108) is not associated with the simple synonym “glioma” and thus, unless a sample for a given glioma sample was described using the string “brain glioma”, which appears to be rare, the MetaSRA failed to annotate this sample as a “brain glioma”. Instead, the MetaSRA labels glioma samples using an alternative “glioma” term from the Experimental Factors Ontology (EFO:0005543), which does not have “brain cancer” as an ancestor term, but instead has “brain neoplasm” as an ancestor (EFO:0003833). This case points to the fact that there is still work to be done in both standardizing the metadata in the SRA and in constructing comprehensive ontologies. Unfortunately, these issues remain out of the scope for this work; however, we now include new text in the Conclusion section that discusses how the original MetaSRA annotations contain some errors and that these errors may propagate to the output of these tools. - Thank you for this suggestion. We have added more detailed instructions for each input parameter. We also perform more thorough input-validation on the user’s input. Lastly, we have added more documentation to each function in utils to help a user who wishes to dive further into the code.Responses to minor issues:1. We apologize for this password issue. Given how few dependencies these notebooks utilize, we decided that Docker is probably overkill for this project and therefore we removed this option altogether. We instead uploaded these notebooks to Google Colab to run in the cloud. If a user would like to run the notebooks locally, we now detail all of the dependencies in the file “requirements.txt” within the repository and offer guidance on installing these dependencies in the README. 2. Thank you for these suggestions. We flipped the barcharts 90 degrees and also use a different color palette for each pie chart. We note that the same samples are used to construct each of the four pie charts.3. We added text to this sentence highlighting another example of a temporal property: time in which cells have spent differentiating in vitro. To this end, we have also added another parameter to the query that enables users to select only in vitro differentiating cells in order to answer possible biological questions pertaining to differentiation.4. This is definitely an important feature, thank you for suggesting it. We now enable the user to match by age and sex in the notebook (see Section “3. Set filtering parameters”) in the notebook. Specifically, in the notebook, if the user sets the variable “MATCH_BY_SEX” to True, we only consider samples that are annotated by sex in the MetaSRA and then match accordingly. Similarly, if the user sets “MATCH_BY_AGE” to True, we only consider samples that are annotated with age and then match accordingly." } ] } ]	1	https://f1000research.com/articles/9-376
https://f1000research.com/articles/9-900/v1	04 Aug 20	{ "type": "Correspondence", "title": "Comment on Lachenmeier et al (2020) “Are side effects of cannabidiol (CBD) products caused by tetrahydrocannabinol (THC) contamination?”: disputation on various points in the publication", "authors": [ "Daniel Kruse", "Bernhard Beitzke", "Daniel Kruse" ], "abstract": "This Correspondence article is a counterstatement to a Brief Report published by Lachenmeier and co-workers on 17th February 2020 in F1000Research: “Are side effects of cannabidiol (CBD) products caused by tetrahydrocannabinol (THC) contamination?”. This counterstatement proposes that the authors of that article neither present proof or evidence for the alleged side effects of CBD products (no case reports presented with utilisable data), nor do they show that side effects are due to the presence of THC. Primarily, there is no clear definition of THC because the authors do not explain whether they mean Delta9-THC only (without its precursor tetrahydrocannabinolic acid (THCA)) or total-THC (the sum of Delta9-THC and its precursor THCA, normalised to THC); indeed EU Recommendation 2016/2115 on the monitoring of cannabinoids in food requires the measurement and documentation of the precursor acids complementary to the decarboxylated cannabinoids. The key part of the authors’ work – Table 2 with the assessment of the CBD products – leaves the reader in the dark about the nature of “THC”. This is all the more concerning because acid-free Delta9-THC is psychotropic but THCA is not. Additionally, the classification of the CBD products (“toxicity assessment”) presented is based on the assignment of the quantitative relation to the LOAEL (lowest observed adverse effect level) of THC (2.5 mg of acid-free Delta9-THC per adult and day as assigned by EFSA, 2015). However, many assumptions by Lachenmeier et al. on daily intake of CBD products are questionable, in particular food supplements, where the recommended daily consumption was missing on the label. Finally, the authors of the paper also compare their findings with the German recommendations on maximum levels of total-THC in food, ignoring that those limits refer to total-THC and the ready-to-eat products, and not to the food ingredient itself – in particular hemp tea products.", "keywords": [ "Cannabidiol", "tetrahydrocannabinol", "hemp", "illegality", "daily intake", "toxicity", "side effects" ], "content": "Introduction\n\nThis Correspondence article was written by members of the European Industrial Hemp Association (EIHA; https://eiha.org/) and EIHA Advisory Committee to highlight their concerns about the Brief Report published by Lachenmeier et al.1 on 17th February 2020 in F1000Research: “Are side effects of cannabidiol (CBD) products caused by tetrahydrocannabinol (THC) contamination?”. The content and argumentation of the Brief Report has alienated our organization in many ways. For this reason, as a European professional association, we feel compelled to comment on this publication and its content.\n\nThe article by Lachenmeier et al. revealed the following:\n\n- purported THC-like side effects aimed to show of CBD or CBD-containing products cannot be sequelae of CBD conversion to THC by gastric fluid but are the effects of the residual THC in such products;\n\n- CBD-containing hemp products (teas, extracts sold as food supplements) give rise to THC-like side effects because in many cases either the LOAEL or the European Acute Reference Dose of THC will be exceeded after consumption of such products;\n\n- the high THC-content of CBD products is a “scandal on the food market”, and food business operators “placed unsafe and approved products on the market”;\n\n- the current regulatory framework were “insufficient to adequately regulate products in the grey area between medicines and food supplements”.\n\n\nAlleged side effects of CBD products\n\nFirstly, it should be noted that, as the authors themselves admit, cases of alleged side effects of products containing CBD claimed in the publication are anecdotal. It is striking that no reference is given as proof of evidence. Instead, the authors try to present findings on effects of Epidiolex® as evidence for their statement on CBD-containing hemp products. Indeed, the reference to rare side effects of the Epidiolex® studies is displaced, especially since Epidiolex® (against special forms of children's epilepsy) is \"pure\" CBD with a much higher dosing regimen for a pharmaceutical/metabolic action and cannot be compared with low dosed CBD oils and hemp extracts with naturally present levels of Cannabinoids. On the contrary, it has been suggested that some adverse effects of CBD in the clinical studies (for Epidiolex®) may relate to interactions with other antiepileptic drugs2. Moreover, an assessment of the drug status of Epidiolex® by the US Public Health Service came to the conclusion: “Thus, it is unlikely that THC contributed to the slight positive responses on some of the subjective measures or contributed to the euphoric [adverse event] responses reported following the higher doses of CBD.”3\n\nHence the question is: have the purported side effects after uptake of CBD oils or hemp extracts been measured and proven? Have there been serious side effects showing that these products were not safe, and which products exactly gave rise to the reported side-effects?\n\nAll this is not explained in the article by Lachenmeier et al, although the title of this publication suggests that these side effects are hard facts. No reference or proof is given for the alleged side effects of the CBD products in question. Furthermore, a statement by the British FSA (FSA 20-01-03)4 states:\n\n\"18. We have not been made aware of any safety incidents relating to CBD products on the market, so we are not planning to insist on an immediate removal of the products from the shelves. That said, it is important that industry puts these products through the authorisation process as the process is there ...\"\n\nThis statement is representative for CBD products all over Europe because the UK was and is an important market for the Food Business Operators (FBOs) on the European continent.\n\n\n\"THC\" definition and estimation of daily dose of products\n\nThe main subject of the article by Lachenmeier et al is \"THC”, which is neither precisely defined in the publication nor correctly explained, i.e. there is no differentiation between Delta9-THC (acid-free) and Delta9-tetrahydrocannabinolic acid (THCA), the latter being the non-psychotropic precursor of THC and not contributing to the \"toxic effects\" of \"THC\"5. From the raw data published in Table 2 of Lachenmeier et al, the type of THC cannot be defined for each case, i.e. whether this should mean total-THC (which is the sum of Delta9-THCA, corrected by the molecular weight factor, and Delta9-THC) or Delta9-THC (acid-free) only.\n\nPursuant to the EU Recommendation 2016/21156 on the monitoring of cannabinoids in food, THC should be differentiated correctly between Delta9-THC and its precursor THCA; their respective concentrations should be measured separately, and the contents of both compounds documented correctly, in particular in a scientific paper.\n\nIn favour of the authors, we will assume here that the \"THC\" measured and mentioned in the publication refers only to Delta9-THC (acid-free), and that the analytical method used for this purpose is able to quantify the latter separately from THCA (note: natural products that have not been heated always contain a part as THCA)7. This is also relevant for the samples containing cannabidiolic acid (CBDA) and/or cannabigerolic acid (CBGA) (5 samples tested), as most probably the main part of their \"THC\"-content consists of THCA, which should not be included in the toxicity assessment based on the Delta9-THC content.\n\nMoreover, the statement in Lachenmeier et al. \"Out of 67 samples, 17 samples (25% of the collective) were exceeding the THC LOAEL [Lowest Observed Adverse Effect Level] and were assessed as harmful to human health…\" is not scientifically correct and cannot be assumed as hard fact. The \"THC\" level is in fact the result of the measurement of the \"THC\" concentration by analysis, while the LOAEL is the lowest amount of a substance, which, when measuring certain effects in animal or human studies, shows just observable adverse effect(s). The missing link between the result from the analysis and the dictum of \"LOAEL has been exceeded\" is the (recommended) daily dose of the specific product together with its measured THC-concentration. These data are only available in the raw data published by Lachenmeier et al.8 which should be shown in the publication itself. It should also be noted that for those products for which a recommended daily dose was missing on the label, the authors made an estimation of these daily doses. In many cases, however, they assumed a very high daily dose, which in our view is far from being practical, as can be illustrated by the following examples:\n\nTea products (hemp flowers or leaves): the authors base their calculation on a \"probable intake of 2 portions/day\" (8 g of tea leaves or flowers per person daily), or they assume a daily consumption for tea products taking into account a worst case scenario, concluding the \"LOAEL may be exceeded\". However, in Table 2 it is stated that the THC-LOAEL is exceeded (marked in red). This is confusing to readers who will be unable to distinguish which statement is correct.\n\nFurthermore, the THC intake for all tea products is calculated by multiplication of the estimated daily portion with the measured THC-content in the hemp tea leaves, hence the authors insinuate the ingestion of 100% of the THC found in the hemp “tea leaves”. This type of calculation is not correct. Firstly, a 100% carry-over of THC into the tea preparation (infusion) has been refuted in the scientific literature9. Secondly, as to the German guidelines on total-THC-content in foodstuff, the THC guidance value for tea beverages refers to the ready-to-eat or ready-to-drink products and not to the hemp “tea leaves” green matter. Among EIHA members, there are FBO's who have been testing hemp leaf infusions in water for years, and these reports are available10. This type of estimation may lead FBOs to wonder why they recommend a daily dose on the label as well as a brewing instruction, if this is then ignored or overruled by an authority in the evaluation of marketability.\n\nSyrup with hemp flower extract: a daily portion of 130 g is assumed (because of a missing recommended dose), thus exceeding European Food Safety Authority (EFSA)'s Acute Reference Dose (ARfD)11. One third of this amount (43 g/d) would usually be an extremely high daily intake of syrup for human consumption in everyday practice and would result in a THC intake below the ARfD (approx. 56 µg/d). This would not be admonished, for example in Austria12. The assignment of a daily dose of 130 g therefore seems to have been arbitrary and possibly results-oriented only.\n\nCannabis shot: a daily dose of 60 g is estimated and assumed, which even for a food supplement is objectively to be regarded as very exceptional and extremely high; this amount results in a THC uptake of only 8 µg/d. This product is admonished by Lachenmeier et al. for exceeding the German guidance for THC-levels in food, even though they are actually not regulated by law let alone made binding. In addition, the German recommendations apply to total-THC (including THC acids) and only for ready-to-eat products (i.e. those which the consumer finally ingests after preparation).\n\nCBD oil: first line in Table 2, the following assumption was made (see footnote 2) about this product: \"No labelling about dosage provided on the label. For this reason the consumption of the whole bottle at once as worst case exposure scenario was assumed.\". In all objectivity, this is exaggerated and not a realistic scenario. First, we can assume that it is well known to the consumer of a food supplement on how to take it. Therefore, the consumer will not take the contents of the entire container or bottle at once. The typical consumer of a food supplement is most likely a well-informed person proactively following a healthy lifestyle, feeling responsible for their own health, and who is therefore willing to purchase products that will help to maintain and to strengthen homeostasis – to stay healthy for a longer lifespan. Second, a food supplement is not a basic food (see legal definition of \"food supplements\" in Art. 2 lit. a of Regulation 2002/46/EC13). Third, if there are food supplements with “CBD oil” on the market without correct labelling (e.g. without a recommended daily intake) this constitutes the wrongdoing of some individual market players and not of the whole industry.\n\nFor some samples with the recommended daily dose missing on the product label (marked with footnote 4, Table 2, in Lachenmeier et al.), the basis for the toxicity assessment is unclear from Table 2, whereas in the raw data there is a daily portion given (e.g. 5.0 g for the “CBD buds” or 8.0 g for “Hemp tea with flowers”) and this portion is used for the daily THC-intake calculation.\n\nConsidering these examples, the data in Table 2, on which the publication is based, are derived from unclear premises on the nature of \"THC\" and the daily dosing of the CBD-containing products. Many parts are arbitrarily based on assumptions that are out of touch with reality, and which we do not consider justified.\n\nAdmittedly, a dosage or recommended daily intake should always be found on the label of food supplements (see Article 8(2) of Regulation 2002/46/EC). The recommended intake that consumers usually find on the packaging of the products should be clear and concise, and it should be explicitly indicated “not to exceed the recommended dose” and \"food supplements should not be used as a substitute for a healthy and varied diet\". However, the many cases in which the respective manufacturers have done this correctly seem to be dismissed by the authors.\n\n\nAssessment of the impact of CBD products\n\nThe assumption stated in the publication by Lachenmeier et al. that \"the adverse effects of commercial CBD-products\" (still to be proven by evidence) \"are based on a low dose effect of THC and not due to effects of CBD itself\" lacks sufficient scientific and comprehensible justification as this is only based on the known toxicological effects of isolated Delta9-THC (precisely synthetic Delta9-THC). The interaction between Delta9-THC and CBD, which has been investigated in many studies, is not taken into account here by the authors. Obviously, however, the scientific community is aware that the effects of “THC” are indeed mitigated by CBD (see e.g. Zuardi et al., 198214). A more recent publication on \"Lower-Risk Cannabis\"15 even recommends with the note: \"Given the evidence of CBD’s attenuating effects on some THC-related outcomes, it is advisable to use cannabis containing high CBD:THC ratios. Evidence Grade: Substantial.\". The mechanisms of this interaction are not fully elucidated yet, however, currently it is acknowledged that CBD is a negative allosteric modulator of the CB1 receptor16. It is surprising that the authors do not mention these well-known scientific findings.\n\n\nAlleged \"illegality\" of all hemp products containing CBD\n\nThe authors further claim: \"Basically all available CBD products based on hemp extract marketed as food or food supplement within the EU are therefore illegally sold\". Although this is a regulatory issue, we think it is necessary to comment on this blanket statement.\n\nA general statement on the non-conformity of all available CBD-products with EU regulations is, in our opinion, outside of the scope of the publication. As stated already many times by jurisdiction, the assessment, for example on compliance with the Novel Food Regulation (EU), is always a case-to-case evaluation by authorities or at court. It must also be emphasized that the European Novel Food Catalogue is legally not binding (see legal disclaimer on the corresponding website of the EU COM17). For court decisions, the entries in the NF Catalogue are just indicators or circumstantial/presumptive evidence. Moreover, the burden of proof for the novelty of a food is up to the person or body who makes this statement, whereas the economic operator(s) bear(s) a so-called secondary burden of proof, if necessary, which, however, must not lead to the reversal of the burden of proof18. Although the Federal Administrative Court in Germany (BVerwG) has not yet had to make an explicit statement on this legal issue, in a decision on 29 January 2010 it already indicated that it has doubts about the burden of proof regulation, which some administrative courts have practised elsewhere to date19.\n\nSince experience shows that the federal courts in Germany coordinate their case-law on one and the same provision among themselves, it can be assumed that the BVerwG will ultimately follow the aforementioned legal interpretation of the Federal Court of Justice. This is also in view of the fact that the European Court of Justice (ECJ) already imposes the burden of proof on the party claiming the existence of a pharmacological effect of a foodstuff in cases of doubt. The BVerwG has already fully endorsed this legal interpretation of the ECJ on the rule on the burden of proof there20.\n\nHence, the ECJ did not impose the burden of proof of the non-existence of a pharmacological effect of a foodstuff on the FBO concerned. Therefore, it is all the more unlikely that the ECJ now should come to a different rule on the burden of proof in the issue of a possible \"novelty\" within the meaning of the Novel Food Regulation and an associated abstract health risk, according to which the FBO were required to prove the non-existence of a \"novelty\". This would be completely contrary to the legal doctrine as well as to the factual system.\n\nWhat an authority can do, of course, is to conclude within its expertise on the non-marketability of a specific product because it is \"unsafe\" for the consumer within the meaning of Article 14(1) and (2) of Regulation (EC) No 178/200221, i.e. injurious to health or unfit for human consumption (and this is the primary realm of the local authority). However, such decisions must also be based on scientific evidence (cf. Article 3 et seq. of Regulation (EC) No 178/2002).\n\n\nValue judgement on food producers of hemp products\n\nLachenmeier et al. also claim that \"In our opinion the systematically high THC content of CBD products is clearly a “scandal” on the food market. Obviously, the manufacturers have – deliberately or in complete ignorance of the legal situation – placed unsafe and unapproved products on the market and thus exposed the consumer to an actually avoidable risk.\"\n\nWe consider this sweeping and polemical value judgement misplaced in a scientific work. Reputable, professional producers of food and food supplements always ensure the quality and safety of their products in all steps of production and also observe legal compliance with European and national regulations. These producers have a well-established quality assurance and control for their products and ensure correct labelling, in particular for correct consumer information with a recommendation on the daily intake of the product.\n\nIn fact, the legal situation is not as clear as purported by the authors, and it cannot be generally stated that all CBD-containing products are Novel Foods under the European Novel Food Regulation. At the end of 1997, even the EU Commission with its Standing 2015/2283 Committee on Foodstuffs had established and confirmed that foods that contain parts of the hemp plant, such as hemp flowers, are not covered by Regulation (EC) No. 258/97 on Novel Foods and Novel Food ingredients22\n\nIn Germany, the Federal Government publicly declared the following in the German Bundestag: “The opinions of the European Commission confirming that foods containing parts of the hemp plant are not novel foods remain valid. However, it cannot be concluded from them that all products of the hemp plant, including isolated individual substances such as cannabinoids or extracts enriched with cannabinoids, would be marketable as food. In addition … it must always be checked whether a product in its composition was used as a foodstuff to any significant extent in the EU before 15 May 1997. Otherwise, as in the case of cannabinoids, it must be considered as a novel food”23 .\n\nThe EIHA has already presented evidence to the European Commission, according to which hemp extracts have been consumed as food for centuries, and that the so-called \"low-THC\" varieties from Cannabis sativa L. (thus “industrial hemp” or “usefulness hemp”) have always contained CBD. At this point, it should be mentioned that especially in these industrial hemp varieties – including those that were listed in the EU catalogue of varieties long before 1997 – the relevant CBD content in relation to THC is very high in comparison to \"high-THC\" cannabis varieties. Therefore, such hemp extracts (e.g. those not enriched with CBD) are traditional food and will not fall under the Novel Food Regulation.\n\nHence, the statement by Lachenmeier et al. that the products are unsafe is another blanket statement and must refer to products only that have been in fact proven unsafe for consumption according to current requirements. However, this publication does not scientifically verify this and presents no proof. Moreover, it has been decided many times by jurisdiction that the decision on the novelty of a food is always based on a case-to-case evaluation of the special product in question.\n\nAdmittedly, there are always so-called \"cowboys\" or \"black sheep\" in the market – as in every industry – disregarding the regulations, trying to make a quick profit with the hype around \"legalisation of cannabis\" and cannabinoids. This phenomenon is unfortunately also evident in cases of questionable quality and insufficient attention to labelling (e.g. missing recommended intake).\n\nHowever, discrimination can also occur from undifferentiated action of the authorities with regard to quality and the legal situation vis-à-vis respectable market participants, which clearly leads to abstruse marketing strategies of so called \"free riders\". Such companies, for example sell plain hemp seed oil through pharmacies at exorbitant prices and suggest to the consumer that it is a particularly effective drug or food supplement24. This situation is also EIHA's concern because this jeopardises the reputation of the hemp industry. However, EIHA, as an association of voluntary members of farmers and producers, can only exert positive influence on its own members, but cannot discipline other unwilling market participants. Therefore, EIHA cannot itself control the market, but could help the authorities to regulate it in a scientifically sound, safe and reasonable way – subject to constructive dialogs and cooperation. EIHA therefore demands clear industry standards that are mandatory for all FBO’s in the sector to ensure compliance with the law, consumer safety and also legal certainty for businesses. Work is underway to harmonise the \"self-regulated\" quality standards of some national interest groups or individual economic operators. The time spent by EIHA and its members as companies in defending themselves against the arbitrariness of individual authorities could be used more efficiently and constructively in mutual dialogue. Unfortunately, earlier offers of exchange and cooperation in Germany, unlike in other European countries, have so far been largely ignored. This should also be mentioned against the background that in the meantime EIHA has decided to carry out extensive toxicology studies on CBD and THC conducted by a third party25.\n\n\nJudgement of the hemp industry in the food sector\n\nIn Lachenmeier et al.’s conclusion, the authors quote that \"Currently CBD users must be aware that they may be ‘participating in one of the largest uncontrolled clinical trials in history’”. EIHA does not accept such an insinuation.\n\nThe quote ‘participating in one of the largest uncontrolled clinical trials in history’ is from a Newsweek article calling for attention to a new supposed scandal (see ref 3326 in Lachenmeier et al.), and is stated by Pal Pacher, an investigator at the National Institutes of Health in the US. It should be noted that this is his own personal opinion and also refers to the situation in the US and not in Europe. In the US, there are different regulations and THC limits for products. It is not known at all to which products with which THC-contents Mr Pacher refers to in his statement. However, since Lachenmeier et al. use this quote in their publication, they are effectively transferring this statement to the whole European hemp industry without any critical review to the situation in Germany and Europe. This statement is not supported by any evidence of accordingly serious incidents or poisoning, which can be attributed to the intake of these so-called \"unsafe\" products (see the UK FSA statement cited (footnote 4)) in Germany or the EU. So, where is the evidence for the purported side effects and products being unsafe?\n\nNotably, EIHA's members and other professional market operators would like to iterate that they do not use their customers as human guinea pigs.\n\n\nProposal for a legal ban on hemp extracts\n\nFinally, the authors of the publication suggest that \"products based on hemp extract with a similar composition could be treated as illegal narcotics, prescription drugs or novel foods in order to resolve conflicting rules in the field of narcotics, drugs and food law\". We feel that this statement is unsubstantiated.\n\nIt is well known that the legal classification of the products offered on the market depends on the composition of the products, on the way they are processed and, above all and primarily, on their objective intended use! Of course, products containing cannabinoids can – depending on their objective intended use – be used for food, food supplements, cosmetic products or as pharmaceuticals as well. It is known and common knowledge that certain food ingredients and substances may be legally sold both as food supplements and as pharmaceuticals, as their use and declaration depends mainly on the dosage of the active ingredient(s) and the method of administration, e.g. the route of administration (oral or intravenous or other) and of course depends on the intended and declared use.\n\nOne of many examples is melatonin, where the low-dose substance is sold as a food supplement and the higher-dose form can be sold as a medicinal product (this is the case in Germany at least). Another example is garlic, which is sold as a food, food supplement and pharmaceutical, depending on its use and dosage form. In this case, the ECJ only a few years ago decided exactly on this juxtaposition of food (food supplement) and medicines. Since the authors of the publication work in this field27, we feel that this should be well known.\n\nIt is also common knowledge that lawful use depends on the definitions and legal provisions of the legislation enacted by the EU (in particular (EC) 178/2002 on the general principles of food law, and, for its delimitation, Directive 2001/83/EC28 on medicinal products for human use) and the case law of the ECJ which completes and complements them. In addition, we have the Directive on Food Supplements 2002/46/EC in the EU, which explicitly permits food supplements with \"nutritional as well as physiological effects\". Most CBD products on the EU market fall into this category and are therefore not allowed to have a \"pharmacological, immunological or metabolic action\" (medicinal products by function pursuant to Medicinal Products Directive), and they are not allowed to claim any diagnostic, curative or therapeutic effect or to present themselves as such (the latter as so-called \"medicinal products by presentation\").\n\nHowever, it is also undeniable that natural CBD in food or food supplements is useful and justified because of its physiological effects (in low doses far from those used medicinally)29 – just like other natural substances – especially in the natural matrix of a hemp extract. Even the World Health Organization reports this in its comprehensive latest study from 2018 on Cannabis and THC30.\n\nTherefore, it is not clear why the above-mentioned legal principles and those developed by case law on their classification as food/ingredient should not apply to products containing hemp. The opinion and demand made by Lachenmeier et al. in their conclusions would ultimately mean a general ban on the current use and marketing of products containing hemp as food ingredients. But there is no plausible reason for this, and certainly no scientifically recognised reason.\n\n\nSummary\n\nWe conclude that the content of the publication by Lachenmeier et al. has fundamental flaws, and, in our opinion, does not meet the expectations of a scientific work that should be based on clear facts and evidence. Accordingly, we would suggest that due to its scientific shortcomings, as outlined above, it cannot be consulted and used without restriction for the objective and correct assessment of concrete facts.\n\nNevertheless, EIHA would like to confirm that unfortunately there are certainly a few products on the market that do not contain the level of cannabinoids stated on the label, or which make unjustified and unauthorised health claims on physiological effects. EIHA therefore supports the creation of clear industry standards that are mandatory for all food operators in this segment to ensure legal compliance and safety for both producers and consumers. By now many FBO's already submit themselves to a \"self-regulated\" quality standard, which is why we are currently trying to harmonise these standards as to scope and values – there are already some proposals on the table and work is in progress.\n\nEIHA is working meticulously with the EU Commission and other industry groups on the concepts of a reliable quality assurance system for industrial hemp products. The corresponding proposals will be presented soon, after which they will be assessed by an EU legislator.\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\nNotes\n\n1 “Are side effects of cannabidiol (CBD) products caused by tetrahydrocannabinol (THC) contamination?” by Lachenmeier et al. in the online Journal F1000Research 2020, 8:1394; Last updated 17 Feb 2020\n\n2 Geffrey et al.: Drug-drug interaction between clobazam and cannabidiol in children with refractory epilepsy, Epilepsia 56(8): 1246–51, 2015\n\n3 U.S. Public Health Service, Dep. of Health and Human Services, Office of the Secretary, Letter to the DEA, dated May 16, 2018; Enclosure: Basis for the recommendation to place Cannabidiol in Schedule V of the Controlled Substances Act., Chapter B.2, page 12 (Residual THC Levels)\n\n4 FSA (2020). ‘Food Standards Agency Board meeting – 21 January 2020’. FSA, 3 January. https://www.food.gov.uk/sites/default/files/media/document/fsa-20-01-03-chief-executives-report-final.pdf\n\n5 Dewey, W.L. 1986. Cannabinoid pharmacology. Pharmacol Rev 38(2):151–178;\n\nG. Moreno-Sanz: Can You Pass the Acid Test ? Critical Review and Novel Therapeutic Perspectives of Δ9-Tetrahydrocannabinolic Acid A, Cannabis Cannabinoid Res. 1.1 (2016).\n\n6 https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A32016H2115\n\n7 If it were total-THC, it would not allow any conclusions on the possible daily intake and thus on reaching or not reaching the LOAEL because the latter has been derived by EFSA on the basis of studies with Delta9-THC [Acid-free] only. Consequently, the Acute Reference Dose (ARfD) derived by EFSA in its scientific opinion only refers to Delta9-THC [acid-free].\n\n8 Lachenmeier, D. W. (2020, April 26). Dataset for “Are side effects of cannabidiol (CBD) products caused by delta9-tetrahydrocannabinol (THC) contamination?” https://doi.org/10.17605/OSF.IO/F7ZXY\n\n9 Hazekamp et al., Cannbis tea revisited: A systematic evaluation of the cannabinoid composition of cannabis tea, J. Ethnopharmacol. 113 (2007) 85–90\n\n10 Test reports are available on request from EIHA.\n\n11 EFSA CONTAM Panel (EFSA Panel on Contaminants in the Food Chain), 2015. Scientific Opinion on the risks for human health related to the presence of tetrahydrocannabinol (THC) in milk and other food of animal origin. EFSA Journal 2015;13(6):4141, 125 pp. doi:10.2903/j.efsa.2015.4141\n\n12 Austria has no recommendations or limit values for maximum levels of THC in foodstuffs. There authorities only determine the conformity with the ARfD for Delta9-THC [acid-free] from the EFSA recommendation.\n\n13 https://eur-lex.europa.eu/legal-content/EN/ALL/?uri=celex%3A32002L0046\n\n14 Zuardi et al.: Action of Cannabidiol on the Anxiety and Other Effects Produced by Δ9-THC in Normal Subjects, Psychopharmacol. 1982(76): 245-50.\n\n15 Fischer et al.: Lower-Risk Cannabis Use Guidelines: A Comprehensive Update of Evidence and Recommendations, Am J Public Health. 2017 August; 107(8): e1–e12, Published online 2017 August. doi: 10.2105/AJPH.2017.303818.\n\n16 Pisanti et al.: Cannabidiol: State of the Art and new challenges for therapeutic applications, Pharmacol. Ther., 2017 (175), 133–50.\n\n17 https://ec.europa.eu/info/legal-notice_en\n\n18 BGH GRUR 2015, 1140; https://dejure.org/dienste/vernetzung/rechtsprechung?Gericht=BGH&Datum=16.04.2015&Aktenzeichen=I%20ZR%2027/14\n\nHegele, Zeitschrift für das gesamte Lebensmittelrecht (ZLR), Germany, 2012, 317, 322 f.\n\n19 BVerwG decision of 29 January 2010, 3 B 84/09\n\nhttps://dejure.org/dienste/vernetzung/rechtsprechung?Gericht=BVerwG&Datum=29.01.2010&Aktenzeichen=3%20B%2084.09\n\n20 BVerwG, judgment of 26 May 2009 - 3 C 5.09\n\nhttps://dejure.org/dienste/vernetzung/rechtsprechung?Gericht=BVerwG&Datum=26.05.2009&Aktenzeichen=3%20C%205.09\n\n21 https://eur-lex.europa.eu/legal-content/EN/ALL/?uri=celex%3A32002R0178\n\n22 Letters from the EU Commission to SonnenHaus ÖKO-Handels GmbH of February 3, 1998, and to Alfredo Dupetit Natural Products of March 3, 1998; https://eiha.org//media/2019/05/EIHA-PRESS-NOTES-EXTRACTS.pdf (see page 4). Copies of the letter are provided on request.\n\nPresentation by M. Reinders: Bringing hemp based products to market, 4th Dec 2019, Cannabis Innovation Hub, London, see charts 16, 17.\n\n23 Statement of the Federal Government in the German Bundestag of 25.07.2019, printed matter 19/11922, p.2.\n\nhttp://dipbt.bundestag.de/extrakt/ba/WP19/2506/250681.html\n\n24 Examples of such companies are known to the EIHA and discussed with the corresponding author.\n\n25 https://eiha.org/2020/06/16/novel-food-pioneering-scientific-studies-commissioned-by-eiha/\n\nhttps://canna-biz.legal/author/cannabizlegal/\n\nhttps://hempindustrydaily.com/european-hemp-group-members-approve-plan-for-cbd-thc-studies/\n\n26 https://www.newsweek.com/2019/09/06/cbd-oil-miracle-drug-science-1456629.html\n\n27 Lachenmeier, Walch: Cannabidiol (CBD): a strong plea for mandatory pre‑marketing approval of food supplements, Journal of Consumer Protection and Food Safety,\n\nhttps://doi.org/10.1007/s00003-020-01281-2\n\n28 https://ec.europa.eu/health/sites/health/files/files/eudralex/vol-1/dir_2001_83_consol_2012/dir_2001_83_cons_2012_en.pdf\n\n29 See e.g. Australian Government, Department of Health, Therapeutic Goods Administration: Safety of low dose cannabidiol, Version 1.0, April 2020.\n\nhttps://www.tga.gov.au/alert/review-safety-low-dose-cannabidiol.\n\nS. Shannon et al. :Cannabidiol in Anxiety and Sleep: A Large Case Series, The Permanente Journal 2019;23:18-041, https://doi.org/10.7812/TPP/18-041\n\n30 WHO Expert Committee on Drug Dependence, Fortieth Meeting, 4–7 June 2018: Cannabidiol (CBD), Critical Review Report: https://www.who.int/medicines/access/controlled-substances/CannabidiolCriticalReview.pdf", "appendix": "" }	[ { "id": "68710", "date": "10 Aug 2020", "name": "Gerhard Nahler", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n21/08/2020: Editorial Note\n\nSince the publication of the peer review report, it has been brought to the editorial team's attention that some Competing Interests had not been declared. As part of F1000Research's editorial policies, reviewers are required to declare any competing interests, including financial competing interests, which may influence their peer review report. We have since confirmed with Dr Nahler that they have the following competing interests, which have been added to their peer review report:\nGerhard Nahler works as independent consultant. Among others, he is consultant of the non-profit NGO “ICANNA”, the EIHA and a number of pharmaceutical industries.\n\nIn order to understand the counterstatement of Kruse and Beitzke (2020) it is necessary to read the article of Lachenmeier et al. (20201) first.\n1) The primary message of Kruse and Beitzke in their Abstract is that no proof for putative side effects/adverse effects caused by “CBD-products” is provided by Lachenmeier et al. (2020), nor that such side effects are caused by THC.\n2) The second point is a lack of clarity about the term “THC” used by Lachenmeier et al. Is it used exclusively for the psychotomimetic delta-9-tetrahydrocannabinol (?), the non-psychotomimetic delta-9-tetrahydrocannabinolic acid (?), for a mixture of both substances such as occurring in hemp tea (?), or also for an analogue, delta-8-tetrahydrocannabinol (?) which is a minor byproduct with lower psychotomimetic properties in hemp extracts?\n3) The third point concerns the “safety” of commercial CBD-products analysed by Lachenmeier et al., in relation to the content of THC. Here, Kruse and Beitzke criticise unrealistic assumptions concerning the calculation of the supposed daily intake of CBD-products and a lack of clarity of the table 2 in the main part of Lachenmeier’s article (concentrations of cannabinoids are communicated by Lachenmeier et al. only as a separate supplemental material; without, the reader cannot verify the assumptions of Lachenmeier et al.).\nAd 1) According to the title, the article of Lachenmeier et al. (2020) focuses on “side effects of cannabidiol (CBD) products” caused by ”contamination” with THC. As CBD products vary widely in their composition beyond CBD as main ingredient, a more detailed characterisation of products, their composition and various other phytocompounds, as well as a comparison to the characteristic side effects of the two pure substances, CBD and delta9-tetrahydrocannabinol (in short THC) would help to better understand the differences. In Lachenmeier’s article, various CBD-products based on hemp extracts or flowers, often consumed as “supplements”, are addressed, therefore containing not only CBD but a number of other phytosubstances, such as delta9-tetrahydrocannabinol (in short THC) among numerous other natural byproducts in varying ratios. The nature and quantities of these other byproducts depend, among many others, on the extraction process and may also include the respective acids CBDA and THCA which have different properties compared to the decarboxylated substances CBD and THC. Lachenmeier’s article focuses therefore on ill characterised “CBD-products”. As a uniform “CBD-product” does not exist, products likely vary in their activity and side effect profile. The statement of Lachenmeier et al. on page 6 “our results provide compelling evidence that THC natively contained in CBD products by contamination may be a direct cause for side effects of these products” is not supported by a listing of such side effects, including their frequencies and/or the respective literature. The title of Lachenmeier’s article assumes that side effects occur with CBD-products and the reader might expect that this will be further addressed in the article; however, this is not the case. The only “side effects” reported read as follows (section “Introduction”): “some pediatric studies in epilepsy patients with orally administered CBD also reported adverse effects such as drowsiness and fatigue that could be explained by pharmacological properties of THC rather than of CBD (8–10).” A further, more detailed description of the nature of side effects supposed to be caused by THC in CBD-(food) products is missing. This is clearly criticised by Kruse and Beitzke. Patients in the articles referenced by Lachenmeier et al. received either extracts (Ref.8, a US parent-survey) or pure CBD (CBD/Epidyolex of GW, purity ~99% with ≤0.1% THC) for treatment-resistant epilepsy and in daily dosages exceeding in some cases 25mg CBD/kg body weight. These subjects received, however, antiepileptic drugs in addition, known to cause, e.g., sedation. Therefore, this can hardly serve as reference for side effects of CBD-products (notably food-products, hemp extracts) taken by a normal, healthy population as is addressed by Kruse and Beitzke. Indisputable differences are therefore the product itself (pure CBD vs. mixtures of phytocompounds), the population (patients vs. healthy individuals) and the intake (regular, high concomitant therapeutic doses vs. low dose, often taken occasionally).\nAlthough this would be of considerable interest, the reviewer is unaware of any systematic evaluation of side effects supposed to be related to CBD-containing, hemp-derived food products. On the contrary, CBD has been described to reduce side effects of THC as has been commented by Kruse and Beitzke. In the article cited (Zuardi et al., 19822) a mixture of CBD with THC (ratio 2:1) attenuated anxiety reactions caused by THC. In hemp, the ratio of CBD:THC is much higher, typically around 20:1; such a modified ratio may also have modified effects which can be further influenced by other phytocompounds. Pure CBD seems to be safe; after excluding studies in childhood epilepsy, the only adverse outcome associated with CBD treatment was diarrhoea (Chesney et al., 20203). In the EFSA report (EFSA, 20154) the lowest observed adverse effect level (LOAEL) of 2.5 mg Δ9-THC/day, corresponding to 0.036 mg Δ9-THC/kg b.w. per day, was derived from “observed central nervous system (CNS) effects (i.e. slight euphoric effects), as well as the increase in heart rate” [according to a more recent review, “a dose of 7.5 mg (THC) did not affect heart rate or blood pressure” (WHO, 20185)]. It has to be reminded that the effect of THC on the heart rate is dose-dependent and transient, and disappears after repeated administration. Whether a slight euphoric effect is “adverse” or not may be disputable and still depends on the situation. Drowsiness or fatigue is uncommon with very low doses of THC.\nAd 2) Although not explicitly stated, “THC” is the psychotomimetic delta-9-tetrahydrocannabinol. If the term is used for “mixtures” this may cause misunderstandings. This may have been the case when Lachenmeier et al. refer to hemp tea, even if it is a common analytical procedure to exhibit the sum of THC + THCA as “THC” for forensic reasons. In addition to request a discriminate use of the term “THC”, Kruse and Beitzke address also in their counterstatement the aspect of an appropriate “labelling” and of mandatory “industry standards” for CBD-products in order to limit a negative impact by “black sheeps” on the reputation of the whole hemp industry. This is highly welcome in the interest of the scientific community, and would be a considerable step forward as it makes discreditation of CBD-products and of the hemp industry less likely; this is clearly encouraged by Kruse and Beitzke. Although not explicitly mentioned as such by Kruse and Beitzke, it is obvious that a correct declaration of the content should not be restricted to the recommended daily consumption and the amount of CBD.\nAd 3) In table 2 of Lachenmeier’s article, all samples for which \"THC > LOAEL“ are marked in red (17 out of 40 samples); of these 17, seven samples) (41%) are “hemp teas”. As this is a notable percentage it deserved a closer look by Kruse and Beitzke. The calculation of the THC-exposure after consumption of tea made from hemp flowers and/or leaves by Lachenmeier et al. is misleading: First, cannabinoids are almost water-insoluble, therefore an extraction of the total amount of THC contained in 8g of dry herbal substance by hot water – as has been assumed in Lachenmeier’s article - is impossible. Even when drug-type cannabis was used for preparation of a tea, the concentration after 20 minutes of boiling (which is not common practice for preparing tea) was 0,043 mg THCA/mL und 0,01mg THC/mL (Hazekamp et al., 20076) or 1.0 to 2.4mg THC per liter according to others (Giroud et al., 19977, cited by Hazekamp et al., 2007). Consumption of two cups of tea, prepared as usual by pouring hot water on dry herbal substance and simmering for about 10 minutes, would normally not result in an uptake of THC exceeding the amount of the lowest observed adverse effect level (LOAEL). This is an important objection made by Kruse and Beitzke. The second, minor overestimation concerns the amount of 8g of dry herbal substance consumed per day. Regular tea bags usually weigh 1.5-2 grams; 8g correspond to 4 to 5 cups of tea per day; this seems to be a rather high consumption, above the average. (The exposure by other CBD-products than hemp tea has not been reviewed).\nWhen Lachenmeier et al. use, in addition to “side effects”, the term “unsafe” for CBD-(food-) products, Kruse and Beitzke request, for the sake of clarity, an explanation why they are “unsafe”, i.e., a description of any, putative or confirmed, short- or long-term negative impact of CBD-products on human health. “Safe” or “unsafe” for food commonly implicates a broader impact on human health than (isolated) side effects. Food can induce, as an example, allergic reactions as side effects still being safe for the large majority of consumers.\nIn short, Kruse and Beitzke clearly describe a number of points in Lachenmeier’s article which would deserve clarification.\n\n) 190267605 180630663 190595270-tea 180776480 190490183-tea 190595273-tea 190595267-tea 190203194 180598182 190495001-tea 190203193 180781746 190400870-tea 180198245 180198246 180598187 190176314-tea (7 of 17 samples); in table 2 of Lachenmeier’s article all samples for which „THC > LOAEL“ are marked in red (17 out of 40 samples); of these 17, seven samples (41%) are “hemp teas”.\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Yes\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? Yes\n\nIs the conclusion balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "5908", "date": "07 Sep 2020", "name": "Dirk W. Lachenmeier", "role": "Reader Comment", "response": "We thank Dr. Nahler for his comments, which mostly refer to our original article1. For this reason, we want to clarify some further points:Ad 1) The evidence regarding side effects was considerably increased since the initial publication of our article, and several studies were added in v31, see also our detailed response2 and our comment to reviewer #2 (Gianpaolo Grassi) above.Ad 2) The issue of EIHA about the definition of THC has also been clarified in v31. We did not use the term for any “mixture” but only for the psychoactive ∆9-tetrahydrocannabinol. However, we wonder about the comment regarding mandatory “industry standards”. How can an industry standard be made mandatory, so that it might resolve the problem of “black sheeps”? They would just similarly ignore the industry standard as they currently ignore the laws and regulations, including those for novel food approval as well as for labelling of food supplements.Ad 3) The questions regarding exposure assessment are answered in our detailed response article2. The question of Dr. Nahler regarding the term “unsafe food” can be answered by looking at the basic food regulation of the European Union:Article 14 of Regulation (EC) No 178/20023 sets out food safety requirements. It requires that food must not be placed on the market if it is unsafe. Food shall be deemed to be unsafe if it is considered to be: i) injurious to health or ii) unfit for human consumption. A criterion commonly applied to determine if a food is deemed unsafe is exceedance of the acute reference dose (ARfD) (see, e.g., Refs. 4-5). A well-founded evaluation of the safety of substances such as THC in foods thus combines exposure calculations based on consumption data and toxicological studies such as the ones found in the scientific opinions of the European Food Safety Authority (EFSA). Our evaluation on a case-by-case basis for each sample based on the EFSA assessment of THC6 showed that typical intake scenarios would lead not only to the exceedance of the ARfD but also to the exceedance of the Lowest Observed Adverse Effect Level (LOAEL) of 2.5 mg/day, which is clearly to be judged as “injurious to health” according to the criteria of Regulation (EC) No 178/20023. It must be mentioned that there appears to be less controversy about lowest observed effect levels of THC than regarding the uncertainty factors to derive ARfD values or acceptable daily intake values (e.g. compare the EFSA position6 to the industry position7).References1 Lachenmeier DW, Habel S, Fischer B et al. Are side effects of cannabidiol (CBD) products caused by tetrahydrocannabinol (THC) contamination? [version 3; peer review: 2 approved, 1 approved with reservations]. F1000Research 2020, 8:1394 (https://doi.org/10.12688/f1000research.19931.3)2 Lachenmeier DW and Walch SG. Evidence for side effects of cannabidiol (CBD) products and their non-conformity on the European food market – response to the European Industrial Hemp Association [version 1; peer review: awaiting peer review]. F1000Research 2020, 9:1051 (https://doi.org/10.12688/f1000research.26045.1)3 European Parliament and Council: Regulation (EC) No 178/2002 of the European Parliament and of the council of 28 January 2002 laying down the general principles and requirements of food law establishing the European Food Safety Authority and laying down procedures in matters of food safety. Off J Europ Comm. 2002; L31: 1–24. Reference Source4 Polinski D, van der Meulen BMJ. Unfit for human consumption. The elusive element in the EU food safety concept of article 14 GFL (April 13, 2019). European Institute for Food Law Working Paper Series 2019/01. Wageningen, The Netherlands (http://dx.doi.org/10.2139/ssrn.3371444)5 Meisterernst A. Gesundheitsschädliche Lebensmittel. In: Lebensmittelrecht. 2020. Verlag C.H. Beck, Munich, Germany (https://doi.org/10.17104/9783406759703-15)6 EFSA Panel on Contaminants in the Food Chain (CONTAM): Scientific opinion on the risks for human health related to the presence of tetrahydrocannabinol (THC) in milk and other food of animal origin. EFSA J. 2015; 13(6): 4141. Publisher Full Text7 Sarmento L, Carus M, Grotenhermen F, Kruse D, Nahler G, Pirich E, Brenneisen R, Grassi G. Scientifically Sound Guidelines for THC in Food in Europe. 2015. Nova-Institute, Hürth, Germany (https://pdfs.semanticscholar.org/117b/267905764ec7cc8bb64a0aa717959130c2df.pdf)" } ] }, { "id": "68709", "date": "24 Aug 2020", "name": "Gianpaolo Grassi", "expertise": [ "Reviewer Expertise Plant breeding", "hemp", "cannabis", "cannabinoids", "foods", "regulation", "THC" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n09/09/2020: Editorial Note\n\nSince the publication of the peer review report, it has been brought to the editorial team's attention that some Competing Interests had not been declared. As part of F1000Research's editorial policies, reviewers are required to declare any competing interests, including financial competing interests, which may influence their peer review report. We have since confirmed with Dr Grassi that they have the following competing interests, which have been added to their peer review report:\nGianpaolo Grassi has the following competing interests:\n1) He contributed to the EIHA sponsored guidelines for THC. 2) He collaborated with Valoya, which led to the development of Canna+ line products. 3) He was an invited speaker at an EIHA conference in 2015.\nFor the above competing interests he has never received money directly, but Valoya has supported projects in the public research institute where he was in charge. EHIA has never paid any consultant fee or contributed in any way to his research activities.\nHe has been retired since last April and confirms these competing interests have not affected his ability to write an objective and unbiased review of the article.\n\nOne of the most abundant problems in the article by Lachenmeier et al. (2020) is confusion. Many of these topics involved are described by previous reviewers and very well by Kruse and Beitzke. I would like to point out the problem of the heterogeneity of the products sampled and tested. They are very different from each other and certainly produced by many companies, in very different conditions, using variable botanical raw materials, of European and non-European origin. The origin adds great confusion to the discussion because if we consider the European hemp varieties we should find in them no more than 0.2% THC and therefore its residues in oil and extracts should be affected by this condition. In non-European varieties we have often found the THC level over 1% (five times the European level) and obviously the THC contamination of oils and extracts will be higher than in products derived from European varieties. Raw materials derived from non-European varieties are widely used in Italy because they are cheaper than European or Italian hemp raw materials (seeds and dried leaves).\nIn Italy, the Ministry of Health has received in previous years only two reports of side effects derived from hemp oil, without the addition of CBD. The reason was that the origin of the seed used to extract the oil was from China and was used as a supplement taken over a long period of time with high daily doses prescribed by the doctor to a child and a young woman.\nA large number of samples tested by Lachenmeier et al. (No. 12) are leaves and flowers used as tea. In this situation the natural cannabinoid content cannot be modified by purification so only the harvest time and the age of the leaves could have a relationship with the THC content which will always be present, but certainly less than 0.2%. Tea preparation involves a high temperature for extraction and this always causes the complete decarboxylation of THC and the activation of its psychotropic effect.\nWhen we consider the samples evaluated by Lachenmeier et al. in the article, they mostly derive from CBD extract which we could find obtained in two main ways: 1. Concentrated cannabinoids (full spectrum) and 2. Purified CBD (99% pure in crystalline form).\nThe most trusted companies use pure CBD derived from crystals dissolved in vegetable oils (sesame, olive, sunflower or hemp seeds). In the case of hemp seed oil, THC contamination is certainly in the 1-5 ppm range and this concentration is admitted by many European countries (Germany, Italy). CBD supplement or foods derived from crystallized non-psychotropic cannabinoids (CBD and CBG) should be identified on the product label so that consumers can be properly informed and could identify a company that describes it correctly the origin of the Product.\nMany of the problems related to the \"THC-like\" side effect could be avoided if the origin of the product or standardization of production could include the block chain procedure or a consortium of manufacturers following a strict production protocol (e.g. see Canadian Manufacturers Test Pledge). It is a consequence of the lack of complete and clear rules and control by third parties (National Authority) because the hemp and cannabis market includes hundreds of products with many origins and the safety of the active ingredients must be guaranteed.\nThe suggestions to the Authorities listed in Kruse and Beitzke's document have been underlined in many countries for years and years, but politics has little interest in regulating the laws and the cannabis market probably because the size of this market is too small or probably because it is interesting for some lobbies.\nHowever, in the paper by Lachenmeier et al. there is a lot of confusion and opinions that are outside the scientific task of such a publication. Confusion is very often the strategy used to limit the ability of consumers and public opinion to make their own choices. In extreme situations, lies add to the confusion and in the case of cannabis this has been done frequently. It should be time for Europe to consider the topic of cannabis correctly and could make an objective choice supported by scientific evidence where opinion or personal orientation as reported in the article by Lachenmeier et al. should be omitted.\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Yes\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? Yes\n\nIs the conclusion balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "5909", "date": "07 Sep 2020", "name": "Dirk W. Lachenmeier", "role": "Reader Comment", "response": "As the reviewer is mostly concerned about our original article1, we hereby want to shortly comment about the following points: Confusion about CBD products on the market A major point of Dr. Grassi appears to be the “confusion” about the commercial market with CBD products, meaning a large heterogeneity of the products sampled and tested in our article1. We actually agree with this assessment, which is also in line with Kruse and Beitzke2, when they speak about “free riders”, “black sheep” or “cowboys” contributing to a substantial part of the market. What we cannot agree with is why it might not be appropriate to describe this “confusing” CBD market, which is just a reality. It should be considered that all the samples described in our article1 were submitted by the responsible food control authorities for evaluation, and the sampling comprised all sectors of the market including internet retailers. Would it not even be in favour of the reputable part of the hemp industry if food control removes the illegal segment of the market, such as the ones using cheaper imported material with unacceptable levels of contamination? Reports of side effects of CBD products We thank Dr. Grassi for sharing the reports about side effects of hempseed oil from China. In v3 of our article1, we have considerably expanded the section about side effects due to the many recent publications about this topic (see also our detailed response3). An additional article was published more recently summarizing poison control center cases (US national data)4. From the total number of cases involving cannabis and synthetic cannabinoids, cannabidiol cases increased from 0% in 2009-2018 to 17% of all cases in 20194. As of August 31, 2020, poison control centers have managed 1,300 cases in 2020 related to cannabidiol5. Inclusion criteria for samples The reviewer is correct that we included all products advertised as “CBD” in our sample. This naturally includes some teas, which are sold with designations such as “CBD flowers”. Regarding risk assessment, it must be considered that we have not even included THC formation from decarboxylation during tea preparation (the samples were analysed without heating). Finally, none of the samples on the German market were based on purified CBD (99% pure in crystalline form) or synthetic CBD but all were “full spectrum hemp extracts”. Tolerated THC levels in Germany The reviewer is incorrect in stating that Germany admits THC contamination in the 1-5 ppm range. As stated in our article1, the German guidance value for foods in general including food supplements is 150 μg total THC/kg (=0.15 ppm). Future strategy for a regulated CBD market First and foremost, we want to refuse the allegations that our article1 is reporting lies, confusion and opinions. Our research might not represent a favourable picture of the current state of the European CBD industry; however, this finding clearly does not invalidate our scientific results, which were in the meantime replicated by other authorities6-7. Nevertheless, we actually agree with Dr. Grassi that we need a regulation of the CBD market along with clear-cut criteria for enforcement. We actually have formulated such a policy need for years and also have similarly concluded in our article1. We also agree with strict rules about labelling (such as identity and property of the used CBD extract) as well as process control on all steps and traceability. Basically, most of these demands are already mandatorily included in European food laws, and the lack of implementing them is another example of the non-compliance of a part of CBD industry.References1 Lachenmeier DW, Habel S, Fischer B et al. Are side effects of cannabidiol (CBD) products caused by tetrahydrocannabinol (THC) contamination? [version 3; peer review: 2 approved, 1 approved with reservations]. F1000Research 2020, 8:1394 (https://doi.org/10.12688/f1000research.19931.3)2 Kruse D and Beitzke B. Comment on Lachenmeier et al (2020) “Are side effects of cannabidiol (CBD) products caused by tetrahydrocannabinol (THC) contamination?”: disputation on various points in the publication [version 1; peer review: 1 approved]. F1000Research 2020, 9:900 (https://doi.org/10.12688/f1000research.25354.1)3 Lachenmeier DW and Walch SG. Evidence for side effects of cannabidiol (CBD) products and their non-conformity on the European food market – response to the European Industrial Hemp Association [version 1; peer review: awaiting peer review]. F1000Research 2020, 9:1051 (https://doi.org/10.12688/f1000research.26045.1)4 Choi NG, Marti CN, DiNitto DM, Baker SD. Cannabis and synthetic cannabinoid poison control center cases among adults aged 50+, 2009–2019. Clinical Toxicology 2020, in press (https://doi.org/10.1080/15563650.2020.1806296)5 National Poison Data System. Track Emerging Hazards. Cannabidiol (CBD) National Poison Data System, American Association of Poison Control Centers. 2020. Reference Source6 FSAI: Consumers being put at risk and misled with some CBD food supplements. Food Safety Authority of Ireland; 2020. Reference Source7 European Commission: Rapid Alert System for Food and Feed (RASFF). Brussels, Belgium. European Commission; 2020. Reference Source" } ] } ]	1	https://f1000research.com/articles/9-900
https://f1000research.com/articles/9-899/v1	04 Aug 20	{ "type": "Systematic Review", "title": "Cardiovascular implications of coronavirus disease 2019: a systematic review", "authors": [ "Ravi Ranjan Pradhan", "Ajay Kumar Yadav", "Shobha Mandal", "Ajay Kumar Yadav", "Shobha Mandal" ], "abstract": "Background: World Health Organization has declared coronavirus disease 2019 (COVID-19) as a Public Health Emergency of International Concern. It has killed thousands and millions are infected worldwide. Though COVID-19 is supposed to be primarily a disease of the respiratory system, it also has widespread implications on other systems as well. The aim of this systematic review is to summarize the cardiovascular implications of COVID-19. Methods: PubMed, PubMed Central, EMBASE, and Google Scholar were searched for peer-reviewed articles which aimed to delineate the cardiovascular implications of COVID-19. Results: A total of six articles (five original articles and one case report) were included. We found diverse cardiovascular implications of COVID-19 ranging from acute cardiac injury to death. New-onset abnormalities in electrocardiogram or echocardiogragram, elevated plasma levels of cardiac troponin, NT-proBNP, and D-dimer have role in early identification of acute cardiac injury in such patients. Additionally, cardiac troponin and NT-proBNP can be used to evaluate prognosis and possible need for intensive care in these patients. Conclusions: Acute cardiac injury is common in patients with COVID-19. Aggressive supportive management based on prognostic indicators along with management of heart failure, arrhythmias, acute coronary syndrome and thrombosis can improve clinical outcomes in such patients.", "keywords": [ "Coronavirus disease 2019", "COVID-19", "Cardiovascular implications", "Acute cardiac injury", "Death" ], "content": "Introduction\n\nIn late 2019, a cluster of cases of ‘pneumonia of unknown origin’ emerged which was linked to seafood wholesale and wet market in Wuhan, China1, that heralded the onset of coronavirus disease (COVID-19). The disease has now spread rapidly to several countries around the globe and has been declared a pandemic by WHO2. By April 8, 2020, a total of 1,441,589 confirmed cases of COVID-19 with mortality of 82,933 were reported2. COVID-19 is probably the greatest threat to mankind of 21st century; it has not only challenged the current medical practices, but also imposed a huge psychological and socio-economic burden to the entire world.\n\nCoronaviruses are a group of pathogen that primarily target the human respiratory system3. However, previous outbreaks of coronaviruses, i.e. severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome (MERS), have already shown multisystem involvement, including of the cardiovascular system, as well4–6. Given the increasing number of confirmed cases and death due to COVID-19, it is important to be acquainted with cardiovascular manifestations induced by this viral infection. However, there is only limited published data pertaining to cardiovascular presentations of COVID-19. The present systematic review aims to describe the cardiovascular implications of COVID-19 and fill the gap in the knowledge regarding understanding of varied cardiovascular manifestations of COVID-19.\n\n\nMethods\n\nWe followed the recommendations established by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement7,8. The systematic review protocol was not registered due to the urgency of the matter and very limited available evidence on the topic.\n\nFor quality assessment, we used the Newcastle-Ottawa scale (NOS). The total score was found to be 9 and all the studies are of high quality9.\n\nPublished peer-reviewed articles from January 1, 2020 until June 15, 2020 which aimed to assess cardiovascular implications in patients with COVID-19 were included. Article language restrictions were not imposed. In addition to original articles, case reports, and case series were also included. Editorials, letters to editor, and correspondences were excluded.\n\nWe searched standard relevant publications indexed in PubMed, PubMed Central (PMC), EMBASE, and Google Scholar. Databases were searched using the search terms under the two search themes below, combined using the Boolean operator “AND”. For COVID-19, we used “Novel coronavirus”, “Novel coronavirus 2019”, “2019 nCoV”, “Coronavirus Disease 2019”, “COVID-19”, “Wuhan coronavirus,” “Wuhan pneumonia,” and “SARS-CoV-2.” For cardiovascular implications, we used “Cardiovascular implications”, “Acute myocardial injury”, “Acute cardiac injury”, “Arrhythmias”, “Heart failure”, and “Myocardial infarction”. A thorough review of the references revealed further relevant articles.\n\nFirst of all, we screened articles by title and abstract. Then, we examined full texts of relevant articles for inclusion and exclusion criteria. A data abstraction spreadsheet using Microsoft Excel version 2013 was developed, which included the following information: author, year of publication, journal, and country where the study was done, study design, sample size, baseline characteristics and laboratory parameters of the patients and cardiovascular implications of COVID-19. A third researcher checked the article list and data abstraction spreadsheet to ensure there were no duplicate articles. Any duplicated article was counted as a single article.\n\n\nResults\n\nA total of 315 articles were retrieved using the search strategy. After screening by abstract and title, 52 articles were selected for full-text assessment. Of these, 46 articles were excluded as they were not relevant to research aims and objectives (Figure 1).\n\nAll the studies were conducted in China in 2020 and all of them were original articles except for one case report, by Cui et al.10. Among the original articles, all were retrospective studies except study by Huang et al.11, which was a prospective, observational study. All the studies have categorized patients into two groups: non-ICU/non-critical/without myocardial injury/with normal cardiac Troponin group and ICU/critical and severe/with myocardial injury/with elevated cardiac Troponin group. Critical and severe COVID-19 was defined as presence of one of the following conditions: respiratory failure requiring mechanical ventilation; shock; failure of other organs that requires treatment in ICU. Wang et al.12 and Huang et al.11 defined acute cardiac injury as blood levels of hypersensitive troponin I above the 99th percentile upper reference limit (>28 pg/mL) or new abnormalities shown on electrocardiography and echocardiography. Xingwei et al.13 defined myocardial injury as blood levels of myocardial troponin ≥3 times the upper reference limit (34.2 ng/L). However, Chen et al.14 and Guo et al.15 defined myocardial injury as serum levels of troponin above the 99th percentile upper reference limit (Table 1).\n\nICU: intensive care unit; NA: not available; nCoV: novel coronavirus; RT-PCR: reverse transcriptase-polymerase chain reaction; WHO: World Health Organization\n\nThe median age of the patients in all the studies was above 50 years, except in the studies by Huang et al.11 (median age, 49 years) and Cui et al.10 (patient’s age; 55 days). In all studies, patients requiring ICU/critical care or patients with myocardial injury/elevated cardiac troponin were older comparatively, except in study conducted by Huang et al.11 (median ages of patients in non-ICU and ICU groups were same). Male predominance was seen in all studies except those by Cui et al.10 and Guo et al.15. All the studies have included the underlying co-morbidities of the patients (Table 2).\n\nICU: Intensive Care Unit\n\nNA: Not available\n\nWang et al.12 established that among the 138 patients, acute cardiac injury was seen in n=10 (7.2%), shock in n=12 (8.7%), and arrhythmia in n=23 (16.7%) patients. ICU patients were more likely to have one of these complications than non-ICU patients (p < 0.001) [Table 3]. ICU patients demonstrated significantly high levels of creatine phosphokinase myocardial band (CPK-MB), cardiac troponin I (cTnI) and D-dimer. Similarly, the number of patients who had procalcitonin levels >0.05 ng/mL was higher in the ICU group compared with the non-ICU group (n=27 [75%] vs n=22 [21.6%]) (Table 4).\n\na Group with myocardial injury\n\nb Group without myocardial injury\n\nc Group with elevated cardiac troponin T\n\nd Group without elevated cardiac troponin T\n\nNA: not available.\n\nCPK-MB: Creatine phosphokinase myocardial band; CPK: Creatine phosphokinase; hsCRP: Hypersensitive C-reactive protein;NT-proBNP: N-terminal-pro brain natriuretic peptide.\n\na Group with myocardial injury\n\nb Group without myocardial injury\n\nc Group with elevated cardiac troponin T\n\nd Group without elevated cardiac troponin T\n\nThe study conducted by Huang et al.11 found that among the 41 patients, acute cardiac injury was seen in n=5(12%) and shock in n=3 (7%) patients. Acute cardiac injury n=4 ([31%] vs n=1 [4%]) and shock (n=3 [23%] vs n=0 [0%]) were seen more commonly in ICU patients compared with non-ICU patients (Table 3). Besides, ICU patients demonstrated significantly high levels of creatine phosphokinase (CPK), and D-dimer. Number of patients who had procalcitonin levels >0.05 ng/mL and cTnI >28 pg/mL (99th percentile) were more in ICU group compared with non-ICU group (Table 4).\n\nSimilarly, Chen et al.14 observed that among the n=150 patients, n=22 (7.1%) had acute cardiac injury. In the same study, out of n=24 patients in the critical group, n=15 (62.5%) had acute cardiac injury and n=7 (5.6%) out of 126 patients in non-critical group had acute cardiac injury and the difference was statistically significant (p-value <0.001) (Table 3). Hypersensitive C-reactive protein (hs-CRP), N-terminal-pro brain natriuretic peptide (NT-proBNP) and cTnI levels of the patients were significantly higher in critical care cases than in mild cases (p-value <0.001) (Table 4). On univariate logistic regression analysis, critical disease status had a significant correlation with age, male gender, elevated NT-proBNP, elevated cTnI, elevated hs-CRP, hypertension, and coronary artery disease (all p<0.05). Multivariate logistic regression analysis revealed that elevated cTnI (OR=26.909, 95%CI 4.086–177.226, p=0.001) and coronary artery disease (OR=16.609, 95%CI 2.288–120.577, p=0.005) were the independent risk factors of critical disease status.\n\nIn study performed by Xingwei et al.13, among the n=54 severe/critically severe patients, acute myocardial injury was discovered in 24 (44.4%) patients while 26 (48.1%) patients died during hospital stay. The in-hospital mortality rate was significantly higher in the myocardial injury group compared to the group without myocardial injury [75.0% (18/24) vs. 26.7% (8/30), p-value = 0.001]. In myocardial injury group, C-reactive protein (CRP) and NT-proBNP levels were also found to be significantly higher than those without myocardial injury (all p-value<0.01)\n\nCui et al.10 described a case report of acute cardiac injury in an infant infected with COVID-19 as suggested by elevated plasma levels of CPK-MB, and cTnI.\n\nGuo et al.15 observed that among the n=187 patients, n=52 (27.8%) exhibited acute cardiac injury as indicated by elevated cardiac troponin T (cTnT) levels. During hospitalization, arrhythmias were seen in n=11 (5.9%) patients. Patient with elevated cTnT levels had more frequent malignant arrhythmias compared with normal cTnT (n=9 [17.3%] vs n=2 [1.5%]). The overall mortality was 23% (43 patients died) (Table 4). The mortality rates were 7.62% (8 of 105), 13.33% (4 of 30), 37.50% (6 of 16), 69.44% (25 of 36) for patients without underlying cardiovascular disease (CVD) and normal cTnT levels, with underlying CVD and normal cTnT levels, without underlying CVD but elevated cTnT levels, and with underlying CVD and elevated cTnT levels, respectively. Elevated cTnT levels was seen more commonly in patients with underlying CVD compared with patients without CVD (36 [54.5%] vs 16 [13.2%]). A significant positive linear correlation of plasma cTnT levels with plasma hsCRP levels (β = 0.530, p< 0.001) and NT-proBNP levels (β = 0.613, p < 0.001) was also observed.\n\n\nDiscussion\n\nIn previous outbreaks of influenza and coronavirus (i.e. MERS and SARS coronavirus), cardiovascular implications like myocarditis, acute myocardial infarction, and acute exacerbation of heart failure (both systolic and diastolic) are well-recognized4,16–19 (Table 5). Based on the studies analyzed, we found that patients with COVID-19 have diverse cardiovascular implications ranging from acute cardiac injury to death. The incidence of acute cardiac injury ranges from 7 to 44% in different studies and the incidence were higher in patients admitted to ICU compared with non-ICU patients (Table 3). Additionally, cardiovascular involvement was more commonly seen in elderly males. Patients with underlying coronary artery disease, hypertension, cardiomyopathy and other co-morbidities are more prone to experience myocardial injury during the course of COVID-19. In these patients, superimposed viral illness damages the myocardial cells by enhancing the systemic inflammatory responses, aggravating hypoxia, and destabilizing coronary plaque. The exact pathophysiology of myocardial injury in previously healthy patients with COVID-19 infection is not fully understood. Cardiac injury can occur via direct or indirect mechanisms (Figure 2). Direct mechanism is exhibited by infiltration of virus into myocardial cells and myocardial inflammation, resulting into cardiomyocyte death. Indirect mechanisms include respiratory failure and hypoxemia, multi-organ dysfunction, hyper-inflammation accompanied by cytokine storm, which ultimately lead to myocardial injury20. Cytokine storm during infection possibly causes a reduction in coronary blood flow, destabilization of coronary plaque and microthrombogenesis21.\n\nSARS: severe acute respiratory syndrome; MERS: Middle East respiratory syndrome; BMJ: British Medical Journal.\n\nInflammatory markers like CRP/hs-CRP, and procalcitonin were found to be significantly elevated in patients with myocardial injury/critical patients/ICU patients; suggesting a role for inflammation in COVID-19-mediated cardiac injury. Increased plasma levels of cardiac troponin, NT-proBNP, and D-dimer or new onset abnormalities in electrocardiogram or echocardiogragram can be helpful in early identification of acute cardiac injury in patients of COVID-19. Besides, cardiac troponin and NT-proBNP may have a role in prognostication and possible need for intensive care in such patients. Aggressive supportive management based on prognostic indicators can improve clinical outcomes in such patients. Management of heart failure, arrhythmias, acute coronary syndrome and thrombosis is important.\n\nOur review has included limited number of available studies with small sample size. Hence, the result of our systematic review should be considered with caution. All studies included in our review were conducted in China and most of them were retrospective. Thus, our review lacks heterogeneous and diverse population. We recommend multicenter, multi-national, prospective studies be carried on cardiovascular implications of COVID-19. It is difficult to postulate myocardial injury as the sole cause of death as it may have occurred due to multi-organ dysfunction.\n\n\nConclusion\n\nAcute cardiac injury is common in patients with COVID-19 and when it occurs, has a poor prognosis. Monitoring of myocardial injury markers and cardiac function is of utmost importance, and attention should be paid to the early identification and comprehensive management of myocardial injury in such patients.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: PRISMA checklist for ‘Cardiovascular implications of coronavirus disease 2019: a systematic review’. https://doi.org/10.6084/m9.figshare.12658409.v18.\n\nThe completed PRISMA checklist is available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\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\nSohrabi C, Alsafi Z, O’Neill N, et al.: World Health Organization declares global emergency: A review of the 2019 novel coronavirus (COVID-19). Int J Surg. 2020; 76: 71–76. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRothan HA, Byrareddy SN: The epidemiology and pathogenesis of coronavirus disease (COVID-19) outbreak. J Autoimmun. 2020; 109: 102433. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYu C, Wong RS, Wu E, et al.: Cardiovascular complications of severe acute respiratory syndrome. Postgrad Med J. 2006; 82(964): 140–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPoutanen SM, Low DE, Henry B, et al.: Identification of severe acute respiratory syndrome in Canada. N Engl J Med. 2003; 348(20): 1995–2005. PubMed Abstract \| Publisher Full Text\n\nKhalid M, Khan B, Al Rabiah F, et al.: Middle Eastern respiratory syndrome corona virus (MERS CoV): case reports from a tertiary care hospital in Saudi Arabia. Ann Saudi Med. 2014; 34(5): 396–400. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoher D, Liberati A, Tetzlaff J, et al.: Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. PLoS Med. 2009; 6(7): e1000097. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPradhan R, Ajay Kumar Y, Shobha M: Cardiovascular implications of coronavirus disease 2019 (COVID-19): a systematic review. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12658409.v1\n\nPeterson J, Welch V, Losos M, et al.: The Newcastle-Ottawa scale (NOS) for assessing the quality of nonrandomised studies in meta-analyses. Ottawa: Ottawa Hospital Research Institute. 2011. Reference Source\n\nCui Y, Tian M, Huang D, et al.: A 55-Day-Old Female Infant Infected With 2019 Novel Coronavirus Disease: Presenting With Pneumonia, Liver Injury, and Heart Damage. J Infect Dis. 2020; 221(11): 1775–1781. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang C, Wang Y, Li X, et al.: Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020; 395(10223): 497–506. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang D, Hu B, Hu C, et al.: Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China. JAMA. 2020; 323(11): 1061–1069. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHe X, Lai J, Cheng J, et al.: [Impact of complicated myocardial injury on the clinical outcome of severe or critically ill COVID-19 patients]. Zhonghua Xin Xue Guan Bing Za Zhi. 2020; 48: E011. PubMed Abstract \| Publisher Full Text\n\nChen C, Yan JT, Zhou N, et al.: [Analysis of myocardial injury in patients with COVID-19 and association between concomitant cardiovascular diseases and severity of COVID-19]. Zhonghua Xin Xue Guan Bing Za Zhi. 2020; 48: E008. PubMed Abstract \| Publisher Full Text\n\nGuo T, Fan Y, Chen M, et al.: Cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019 (COVID-19). JAMA Cardiol. 2020; 5(7): 1–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNguyen JL, Yang W, Ito K, et al.: Seasonal influenza infections and cardiovascular disease mortality. JAMA Cardiol. 2016; 1(3): 274–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPan S, Zhang H, Li C, et al.: [Cardiac arrest in severe acute respiratory syndrome: analysis of 15 cases]. Zhonghua Jie He He Hu Xi Za Zhi. 2003; 26(10): 602–5. PubMed Abstract\n\nLi SSl, Cheng Cw, Fu Cl, et al.: Left ventricular performance in patients with severe acute respiratory syndrome: a 30-day echocardiographic follow-up study. Circulation. 2003; 108(15): 1798–803. PubMed Abstract \| Publisher Full Text\n\nAlhogbani T: Acute myocarditis associated with novel Middle East respiratory syndrome coronavirus. Ann Saudi Med. 2016; 36(1): 78–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAkhmerov A, Marbán E: COVID-19 and the Heart. Circ Res. 2020; 126(10): 1443–55. PubMed Abstract \| Publisher Full Text\n\nHuang C, Wang Y, Li X, et al.: Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020; 395(10223): 497–506. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "70363", "date": "14 Sep 2020", "name": "Siddappa N. Byrareddy", "expertise": [ "Reviewer Expertise SARS-CoV-2", "Cardiovascular diseases", "COVID-19." ], "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\nPradhan et al. used six articles to review the idea that cardiovascular issues can predict the severity of a COVID-19 diagnosis, or that COVID-19 infection can lead to heart conditions. Major strengths of the this review is authors clearly said in discussion that very small number of papers to make clear conclusions. They also used several evidences to convenience readers. However, this manuscript has several weakness and need to be addressed.\n\nOn page four in the section “cardiovascular complications associated with COVID-19 patients”, it mentions that patients admitted to the ICU had a higher rate of cardiovascular complications but doesn’t give a number to that stat until the very end. Will be good to say it upfront.\n\nAlso in the same paragraph, authors mention lot of chemicals like “procalcitonin levels, NT-proBNP”. Would it make sense to describe in more detail?\n\nIn discussion, it would make more sense to have two separate sections detailing patients with COVID-19 who developed heart conditions vs patients with underlying heart conditions who became infected with COVID-19?\n\nThe article Cui et al. describing the infant with COVID-19 and myocardial injury, but it got missed and no further discussion on that and hardly referenced in the actual review.\n\nIt is bit misleading the first sentence of their conclusion is indicative of what they wrote about “Acute cardiac injury is common in patients with COVID-19 and when it occurs, has a poor prognosis\". Because most of the paper was dedicated to people already suffering from cardiovascular issues before being infected with COVID-19\n\nIt will be good to mention section of myocardial injury in younger populations?\n\nAdding more papers and including some mechanistic angle is needed in order to strengthen the review.\n\nThere are several papers published on this topic, therefore it is advised to include better key words and perform better systematic review.\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? Partly", "responses": [] }, { "id": "90989", "date": "11 Aug 2021", "name": "Sanjeet Singh Avtaar Singh", "expertise": [ "Reviewer Expertise Cardiothoracic Surgery", "Mechanical Circulatory Support" ], "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\nMany thanks for the review. This was an interesting read but there are a few niggling issues.\nAbstract:\nReads well but I think 'global pandemic' would be an all encompassing term vs 'Public Health Emergency of International Concern'.\n\nIntroduction:\n\nThe study is slightly dated in that C-19 has affected more than 7 Countries!\n\nThe stats about deaths and mortality should be updated as well\n\nCoronaviruses are a group of PATHOGENS\n\n.... i.e. severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome (MERS), have already shown multisystem involvement, including of the cardiovascular system, as well- the as well can be deleted as its use is improper\n\nThe present systematic review aims to describe the cardiovascular implications of COVID-19 and fill the gap in the knowledge regarding THE understanding of varied cardiovascular manifestations of COVID-19.\n\nDiscussion:\nManagement of heart failure, arrhythmias, acute coronary syndrome and thrombosis is important- this is a redundant statement. If the authors want to keep this, I would cite the reference.\n\nThe manuscript would a further grammatical check prior to submission as there are multiple grammatical errors.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-899
https://f1000research.com/articles/9-894/v1	04 Aug 20	{ "type": "Study Protocol", "title": "Preserving the endothelial glycocalyx in patients undergoing cardiopulmonary bypass: a prospective randomised interventional pilot study of lidocaine and doxycycline (LiDEG trial)", "authors": [ "Adrian Pannekoek", "Mark Johnson", "Donal Buggy", "Warren Pavey", "Adrian Pannekoek", "Donal Buggy", "Warren Pavey" ], "abstract": "Background: During major surgery, particularly heart surgery, an element of the lining of blood vessels, known as the endothelial glycocalyx (EG), can be damaged. This can lead to swelling, low oxygen levels, kidney failure and other problems, which delay recovery. There are laboratory studies that show lidocaine (a local anaesthetic) and doxycycline (an antibiotic) may help protect this lining. The study agents are widely available, cheap and safe drugs. Trial design and objective: This is a phase IV, single centre, prospective, unblinded, randomised, parallel-group trial. The objectives of the trial are to investigate the role of doxycycline and lidocaine as potential agents to reduce EG shedding and correlate with early postoperative outcomes. Methods: 60 adult patients undergoing heart surgery requiring cardiopulmonary bypass (CPB) will be randomly assigned to one of three groups: doxycycline group (oral doxycycline, 200mg preoperatively); lidocaine group (perioperative intravenous lidocaine, 1.5mg/kg bolus at induction followed by 2mg/kg/hr infusion for the duration of surgery); and control group (standard care). The randomisation will be undertaken using a sealed opaque envelope method. The primary outcome will be the relative difference in the biochemical marker of EG injury, syndecan-1, at different timepoints in the intraoperative and early post-operative period. Secondary endpoints include vasopressor requirements, markers of organ dysfunction (lung, kidney, brain, arrhythmia), coagulation and inflammation. Discussion: EG injury is ubiquitous in patients undergoing CPB. Maintaining homeostasis of this delicate layer would appear to be a valuable therapeutic target. To date no agents have been shown to be effective in protecting the EG. Our study agents have shown some promise in the preclinical setting and would represent a novel therapeutic approach should they show a protective effect. Trial registration: Australian New Zealand Clinical Trials Registry, ACTRN12619000621112 (26th April 2019).", "keywords": [ "Endothelial glycocalyx", "lidocaine", "doxycycline", "cardiopulmonary bypass", "anaesthesia" ], "content": "Introduction\n\nHeart surgery is known to have harmful effects on the lining of blood vessels called the “endothelial glycocalyx” (EG)1,2. The EG is a proteoglycan coating on the luminal side of blood vessels, and is made up of a dynamic layer of polysaccharides, the composition of which is balanced under normal physiological conditions by vascular sheer forces and continued biosynthesis. Under normal conditions, the EG provides protection that helps preserve barrier function, modulate inflammation and house anticoagulant factors1. The EG is also important for normal vascular functions, including fluid and protein extravasation, coagulation/haemostasis and microvascular blood flow.\n\nDamage to the EG results in vascular permeability, white blood cell adhesion, changes in coagulation and ultimately organ oedema and dysfunction3, making it a viable causal mechanism for these outcomes noted in post-operative cardiac patients. Supporting this, there is good evidence that the EG is rapidly shed under both inflammatory and ischaemic conditions4. Rehm and colleagues also detected a component of the EG, syndecan-1, in the plasma of patients undergoing procedures requiring local systemic ischemia, including total circulatory arrest and aortic cross clamping, which indicates that shedding has occurred5. Strategies to protect the EG are thus a viable clinical target in order to reduce adverse outcomes. Currently no measures are taken in routine practice specifically to protect the EG.\n\nOne potential strategy is the use of doxycycline, a broad-spectrum matrix metalloprotease inhibitor that reduces metalloprotease activity known to be involved in the EG shedding process4. Research trials have demonstrated that administration of doxycycline reduces EG shedding in rat mesentery vasculature4.\n\nAlternatively, lidocaine is an amide local anaesthetic that has a broad range of applications, including as part of adenosine/lidocaine/magnesium (ALM), a cardiac protective agent. As a combination, ALM is implicated in maintaining endothelium function and vascular permeability and leads to the restoration of EG after shedding6. The primary mechanism within ALM is not yet known, however lidocaine alone has been shown to improve coagulopathies, have vasoactive effects, be a free radical scavenger and have anti-inflammatory properties and is a coronary vasodilator6. Therefore, it is suspected that lidocaine alone may be responsible for the EG-related effects of ALM. In a porcine study, an intraoperative lidocaine infusion significantly reduced the shedding of the EG in a lung transplant model7.\n\nThis research trial aims to investigate EG shedding as a cause of adverse outcomes and evaluate the use of both doxycycline and lidocaine as agents to reduce EG shedding and thereby improve early postoperative outcomes.\n\n1. To evaluate the EG shedding in plasma and coronary sinus effluent at key time points in cardiopulmonary bypass surgery.\n\n2. To evaluate the relative reduction in EG shedding with doxycycline and lidocaine as individual agents.\n\n3. To correlate any reduction in EG shedding to early postoperative clinical outcomes.\n\nWe hypothesize that doxycycline and/ or lidocaine will reduce endothelial glycocalyx shedding in patients during cardiopulmonary bypass surgery.\n\n\nProtocol\n\nThis is protocol version 2.2.\n\nThis trial is a phase IV, single centre, prospective, unblinded, randomised, parallel-group trial consisting of three equal treatment arms (1:1:1): control group, doxycycline group, and lidocaine group. There will be 20 patients in each arm.\n\nThis is a single centre pilot trial that will take place at the Fiona Stanley Hospital (FSH; Perth, WA, Australia), which is a large academic hospital.\n\nAll patients undergoing cardiopulmonary bypass at Fiona Stanley Hospital who are ≥18 years old and provide written informed consent will be eligible to participate in the trial.\n\nPatients will be excluded for the following reasons: patient refusal, allergy to trial drug(s), undergoing heart and lung transplant, doxycycline and/or lidocaine contraindicated in patient management, and history of chronic alcohol abuse.\n\nPatients will be recruited via medical personnel in cardiothoracic and anaesthetic clinics and on the ward when applicable.\n\nWritten informed consent will be obtained at recruitment (Extended data8). This consent will be obtained by trained research nurses, anaesthetists or cardiothoracic surgery staff. Information sheets and consent forms will only be available in English.\n\nParticipants are able to withdraw at any stage of the trial. Samples specifically collected for the trial will be removed and disposed of if requested by the patient. Samples will not be entered to a biorepository.\n\nOutside of initial recruitment, and the possibility of ingesting a single tablet (see Intervention section), participant responsibilities are minimal, and retention should therefore be high with recruited patients.\n\nThe primary outcome is the relative difference in the biochemical marker of EG injury, syndecan-1, at different timepoints in the intraoperative and early post-operative period.\n\nSecondary outcomes include:\n\nVasopressor/inotropic requirements: requirement and agent when leaving theatre (low cut off threshold of 5mcg/minute Noradrenaline); requirement at 6 hours into ICU stay\n\nMarkers of organ function: kidney – Δ creatinine, (Including subgroup analysis for pre-existing renal failure and might need to subgroup into existing and not pre-existing renal failure, requirement of RRT); oxygen requirement – PaO2/FiO2 ratio.\n\nInflammation: white cell count, c-reactive protein, cytokine levels (including tumour necrosis factor α and interleukin (IL)-2, IL-4, IL-6, IL-10, and IL-17 and interferon-γ)\n\nBleeding: blood products usage, ROTEM, prolonged coagulopathy\n\nPatient-based: length of ICU stay and pain severity\n\nUnplanned return to theatre\n\nPostoperative delirium\n\nArrhythmia (presence or absence of atrial fibrillation at 30 days)\n\nWe plan to recruit 60 patients randomised into three groups of 20 (see Figure 1). All recruits will be at FSH. It is difficult to do accurate power calculations as this is an exploratory pilot study, but using non-interventional human data and a published pig model2,7, with data only available for lidocaine, syndecan-1 levels peak at the end of surgery with a mean of approx. 50 pg/mL (but widely distributed), a pig model showed levels reduced by over 50% with lidocaine exposure. Therefore, if we take a mean of 50, with a standard deviation of 25 and anticipate a reduction by 50%, alpha error 0.05 and power of 80%, we require 16 in each group. Since these are very crude figures, we hope to enrol 20 patients per group to increase our chance of seeing meaningful data.\n\nPrevious experience in our department shows a recruitment rate to any clinical trial at approx. 20% of all eligible patients.\n\nThis trial aims to recruit 60 patients. Patients will be screened for eligibility in the preoperative clinic or inpatients seen on the ward. Informed consent will be sought from eligible patients. This will be undertaken by anaesthetic staff or research nursing staff. No additional visits will be required by the participants. Participants will be randomised via blocked number draw and assigned to one arm of the study. Patients will undergo randomisation in groups of three, i.e. at the time of recruitment each patient will be randomised to one of the three trial arms with the process repeating with each subsequent three patients. Randomisation will be conducted by opaque envelope method in a 1:1:1 ratio. The envelopes will be sequentially numbered, and each patient will be assigned to the next envelope. The sequencing will be performed externally to the investigators by the hospital pharmacy (clinical trials) unit.\n\nClinicians and researchers will not be blinded to allocations; however, laboratory staff will not be aware of the allocations and statistical analysis will be performed on the data by researchers on anonymised data blinded to the allocations. Emergency unblinding therefore will not be an issue.\n\nDrugs will be supplied by the South Metropolitan Service Pharmacy Department. All treatments will take place in FSH perioperative department. Treatment conditions will be as follows:\n\n1. Doxycycline group: preoperative doxycycline 200mg to be administered at least two hours before patients go to theatre; in addition, patients are to be sitting upright when taking a doxycycline tablet with appropriate amount of water.\n\n2. Lidocaine group: perioperative lidocaine infusion (1.5 mg/kg bolus given at induction of anaesthesia followed by 2 mg/kg/hr infusion until leaving the operating theatre).\n\n3. Control group: standard care\n\nThe doxycycline group will be administered their treatment in the ward by ward nursing staff. The lidocaine group will be administered by the attending anaesthetist using the BD Alaris PK syringe pump (CareFusion, Switzerland) using a preprogramed protocol of 1.5mg/kg bolus delivered over five minutes followed by 2mg/kg/hr. Outside of the study drugs, concomitant care will be at the discretion of the primary anaesthetist. This is highly standardised in FSH; all patients receive high dose fentanyl, propofol and vecuronium at induction, and blood pressure support with noradrenaline and glyceryl trinitrate. Dobutamine is the inotrope of choice in FSH. Anaesthesia is maintained using sevoflurane and is titrated to Bispectral Index. Patients are anticoagulated with heparin with target activated clotting time of 400 seconds or greater for the duration of cardiopulmonary bypass and reversed with a suitable dose of protamine. All patients receive 2 grams of tranexamic acid at induction.\n\nTrial specific blood samples will be collected intra-operatively and post operation (Table 1).\n\nBlood samples will be collected for processing of plasma and storage for batch analysis by specifically appointed trial staff in anaesthesia and ICU. All samples, with the exception of syndecan-1 will be obtained from the existing hospital computer systems as appropriate. Syndecan- 1 and cytokines associated with EG injury will be quantified by ELISA using commercially available kits. ROTEM after protamine will be performed for all patients.\n\nAll other data will be extracted from the electronic medical record including ICU records. Telephone contact will be established with the participants if required.\n\nA Data Safety Monitoring Board (DSMB) has been set up specifically for this trial. It consists of a multidisciplinary group to review, at regular intervals, accumulating trial data, in order to monitor the progress of this clinical trial. Their role is to provide advice on safety and/or trial conduct issues by making recommendations to the investigators, on whether to continue, modify or stop this trial for safety or ethical reasons. The DSMB consists of three suitable members with no vested interest into the outcome of this trial who have relevant scientific and medical expertise.\n\nDoxycycline and lidocaine are widely available and are used frequently in the both the community and hospital setting with a well-established therapeutic and safety profile. Furthermore, lidocaine is used often in the setting of cardiac surgery for its class 1b antiarrhythmic and analgesic properties.\n\nAny suspected adverse effects, including allergic reactions, due to the study drugs will be reported as an adverse event and investigated appropriately.\n\nBoth the actual and potential risk to patients from both agents used in this trial is considered intermediate.\n\nClinicians administering treatments and taking samples will be given an education session and supervised by investigators initially.\n\nData analysis. Data will be analysed using GraphPad statistical software version 8.4.2 (Prism Software, San Diego, USA).\n\nWe will perform interim analysis after four patients from each group (i.e. 12 patients) and at the halfway point (30 patients) and re look at power analysis if indicated by any early signals or lack of. Patients will be randomised by block number draw whereby participants are randomly assigned to one of the three trial arms in 20 rounds of three, so as to ensure balance across conditions and effective interim analysis. We will perform per protocol and intention to treat analysis on the data. Given the nature of the clinician delivered interventions, we anticipate that very few patients will crossover or be lost to follow-up, therefore the analyses should line up closely.\n\nThe principal investigators have a strong working knowledge of medical statistics; however appropriate statistical specialist input will be sought and utilised for this study. Prior studies have shown non normal distribution of syndecan-1 level distribution between participants required logarithmic transformation to use parametric testing2. We will we therefore scrutinise the data using the D’Agostino & Pearson Omnibus test and suitable parametric or non-parametric statistical tests will be used on the data. Results will be expressed accordingly.\n\nContinuous variables and binary data will be scrutinised using appropriate tests, i.e. chi-squared test for binary outcomes, and Student’s T-test for continuous outcomes.\n\nData management. Information will be stored and analysed on password protected hospital computers at FSH for 15 years. Participants will be anonymised. Demographics, type of surgery etc. and treatment arm will be recorded with the recorded results. All data will be included if possible. Any missing or excluded data will be clearly outlined and explained in resulting manuscript.\n\nAll principal investigators will have access to cleaned data sets.\n\nThe principal investigators, will permit trial-related monitoring, audits, and regulatory inspections, providing direct access to source data/documents. This may include, but not limited to, the DSMB, review by external sponsors, Human Research Ethics Committees and institutional governance review bodies.\n\nFormal systematic auditing and monitoring will be undertaken by the authors under the supervision of the DSMB at regular time points (i.e. after 12 & 30 participants and at the end of the data collection). This study will be undertaken by experienced researchers. If there is evidence of harm at any timepoint the trial will be suspended and investigated by the DSMB and may be prematurely terminated at any timepoint. Being a pilot, the trial will not be analysed for futility at the interim stages.\n\nThe principal investigators state that the trial will be conducted in compliance with the protocol, Good Clinical Practice and the application regulatory requirements. Researchers will regularly liaise with clinicians to further ensure protocol adherence.\n\nData will be systematically collected by experienced researchers. Of note, there is no financial incentive (or any other form of incentive) for the results to show superiority or non-inferiority in this pilot study.\n\nThis project has been reviewed and approved by the health research ethics committee (HREC) of the South Metropolitan Health Service, Western Australia (ref RGS-1190). No atypical ethical or consent issues are associated with this pilot study. Participant Information and Consent Forms will be provided to all participants and are available as Extended data8.\n\nAny modifications with a significant impact on the study conduct, patient safety, design, sample size will require a formal protocol amendment. Significant changes considered necessary by principal investigators will require approval from South Metropolitan HREC and relevant health authorities before being instigated. Administrative changes and minor corrections deemed necessary by the principal investigators will be documented and the HREC notified.\n\nPatients enrolled in the trial are covered by FSH hospital indemnity. Due to the short duration of the intervention and the pharmacokinetics of the study agents, post-trial care is not predicted to deviate from standard of care but will be covered by FSH indemnity if required.\n\n\nDissemination\n\nIt is intended that the results of this pilot study will be published and disseminated in an international journal. A minimum time will be taken to compile results after recruitment of the final participant. Deidentified data will be accessible with the final manuscript.\n\n\nStudy status\n\nThe trial started on 1st May 2020 and is currently recruiting patients. The trial is anticipated to finish on the 31st January 2021.\n\n\nData availability\n\nNo data are associated with this article.\n\nOpen Science Framework: LiDEG, https://doi.org/10.17605/OSF.IO/Y2F8C8.\n\nThis project contains the following extended data:\n\n- Informed consent form\n\nOpen Science Framework: SPIRIT checklist for ‘Preserving the endothelial glycocalyx in patients undergoing cardiopulmonary bypass: a prospective randomised interventional pilot study of lidocaine and doxycycline (LiDEG trial)’, https://doi.org/10.17605/OSF.IO/Y2F8C8.\n\nData are available under the terms of the http://creativecommons.org/publicdomain/zero/1.0/ (CC0 1.0 Public domain dedication).", "appendix": "References\n\nBrettner F, von Dossow V, Chappell D: The endothelial glycocalyx and perioperative lung injury. Curr Opin Anaesthesiol. 2017; 30(1): 36–41. PubMed Abstract \| Publisher Full Text\n\nPesonen E, Passov A, Andersson S, et al.: Glycocalyx Degradation and Inflammation in Cardiac Surgery. J Cardiothorac Vasc Anesth. 2019; 33(2): 341–345. PubMed Abstract \| Publisher Full Text\n\nAnnecke T, Fischer J, Hartmann H, et al.: Shedding of the coronary endothelial glycocalyx: effects of hypoxia/reoxygenation vs ischaemia/reperfusion. Br J Anaesth. 2011; 107(5): 679–86. PubMed Abstract \| Publisher Full Text\n\nMulivor AW, Lipowsky HH: Inhibition of glycan shedding and leukocyte-endothelial adhesion in postcapillary venules by suppression of matrixmetalloprotease activity with doxycycline. Microcirculation. 2009; 16(8): 657–66. PubMed Abstract \| Publisher Full Text\n\nRehm M, Bruegger D, Christ F, et al.: Shedding of the endothelial glycocalyx in patients undergoing major vascular surgery with global and regional ischemia. Circulation. 2007; 116(17): 1896–906. PubMed Abstract \| Publisher Full Text\n\nDobson GP, Letson HL: Adenosine, lidocaine, and Mg2+ (ALM): From cardiac surgery to combat casualty care--Teaching old drugs new tricks. J Trauma Acute Care Surg. 2016; 80(1): 135–45. PubMed Abstract \| Publisher Full Text\n\nRancan L, Simón C, Sánchez Pedrosa G, et al.: Glycocalyx Degradation after Pulmonary Transplantation Surgery. Eur Surg Res. 2018; 59(3–4): 115–125. PubMed Abstract \| Publisher Full Text\n\nJohnson M: LiDEG. 2020. http://www.doi.org/10.17605/OSF.IO/Y2F8C" }	[ { "id": "68613", "date": "05 Aug 2020", "name": "Eero Pesonen", "expertise": [ "Reviewer Expertise Inflammation", "coagulation and related phenomena (including also the endothelial glycocalyx) in cardiac surgery", "solid organ transplantations and sepsis." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMain Considerations\nThe aim of the intended RCT is to investigate the efficacy of p.o. doxycycline and i.v. lidocaine in reducing endothelial degradation (measured as plasma syndecan-1) in heart surgery with CPB. The focus of the RCT is important.\nThe study is already recruiting patients. I am deeply worried of the statistical power of the RCT. I am afraid that it will be underpowered. 1) I suggest revised power calculation after recruitment of 30 patients. 2) To reduce variability, I suggest to exclude certain types of cardiac surgery. Furthermore, I suggest to exclude patients with such medications which may directly influence on the CPB-induced inflammatory reaction, and patients with bleeding diathesis. While these measures reduce variability, they may increase potential intervention effect and thus study power. Above-mentioned measures can be applied although the patient recruitment has already started.\nI do not understand why the authors do not conduct the RCT as double-blind. It would be easy to do and would substantially improve the quality of the study. While the patient recruitment has already started, this modification probably should not be done anymore.\nOtherwise, I believe that the modifications suggested below can be applied although the patient recruitment has already started. However, an approval of the ethics committee may be required.\nAustralian New Zealand clinical trials registry\nThere are numerous and significant discrepancies between the present manuscript and the registry. These should be corrected either in the registry or the manuscript to correspond the actual conduct of the RCT.\nInclusion and exclusion criteria\nIn terms of surgery with CPB, only transplantations will be excluded. Consequently, patients undergoing surgery of the aortic arch (requiring deep hypothermia with/without circulatory arrest or selective cerebral perfusion), descending aorta (spinal cord ischemia) and redo congenital defects (often complex and unpredictable surgery) will be recruited. This will result significant variability of the surgical insult. I suggest to exclude these types of surgery. Most probably, the number of these patients would be low anyway.\nFurthermore, I suggest to exclude patients with preoperative or perioperative corticosteroids or immunomodulatory drugs or any other drugs with significant anti-inflammatory action. Perioperative corticosteroids should not be allowed.\nPatients with bleeding diathesis should be excluded.\nPrimary Outcome\nAccording to the registry, syndecan-1 concentration will be expressed as a fold-increase of baseline (pre-induction) level. This should be indicated also in the manuscript.\nI support the use of fold-increase to manage the vast inter-individual variation of absolute syndecan-1 plasma concentrations. However, even fold-increase is not without problems. If the preoperative level is small, it will yield high fold-increase even for small absolute increase and vice versa. Also fold-increase can result in large variation. I suggest to calculate the primary outcome both as fold-increase and as absolute concentrations. If so, this should be indicated in the manuscript. This is justified because of vast inter-individual variation of absolute syndecan-1 plasma concentrations.\nSecondary Outcomes\nThe secondary outcomes (how and when they are measured and calculated?) should be defined unequivocally.\nVasopressor/inotropic requirements: Will some vasopressor and inotropic score be calculated? There are formulas to calculate equivalent doses? (Later in the description of patient care: How realistic it is that only noradrenaline and dobutamine will be used?)\nMarkers of organ function: Delta-creatinine, during how many postoperative days? P/F-ratio, at what time points or when?\nBleeding: Blood products usage, the length of the postoperative observation period? Prolonged coagulopathy, definition?\nROTEM, which parameters will be used?\nPain severity, how do you measure?\nPostoperative delirium, definition?\nSample size\nI assume that the sample size was based on unpaired t-test? The test applied for the sample size calculation should be indicated. To be precise, the power calculation should be based on repeated measures ANOVA. Because there is paucity of applicable literature, in my opinion, unpaired t-test is justified. After all, power calculation is always only estimation.\nIt should be clearly indicated, that the power calculation applies only between the control group and one of the intervention groups (doxycycline or lidocaine). The assumed effect does not apply for comparison between the intervention groups.\nThe estimated reduction of 50% is high and possibly unrealistic. Furthermore, the assumed SD is significantly lower than in the syndecan-1 results of the reference #2. I am afraid that the power calculation is too optimistic. On the other hand, the sample size is now calculated for actual syndecan-1 plasma concentrations. To reduce the effect of large inter-individual variation in plasma syndecan-1 concentrations, the authors will calculate fold-increase (i.e. multiples of the respective baseline values), which I fully support. It might be better to conduct the power calculation also for fold-increase values. The authors should screen the literature if they could fine applicable data. In my opinion, it would be justified to choose the power calculation (absolute concentrations or fold-increases), which yields lower patient number.\nFinally, I suggest a revised power calculation as an interim analysis (see below).\nBlinding\nI do not understand why the authors do not conduct the study as double-blind? It would have been easy to do with placebo tablets and saline syringes with non-transparent foil. In all groups, the patient would receive a tablet (either doxycycline or placebo) and infusion (either lidocaine or saline). While doxycycline and lidocaine are well-known drugs, there should be no ethical concerns, either. Double-blind design would improve the study substantially.\nProtamine Dose\nDoes the study site not have a more or less established protocol for protamine dosing (initial and potential additional doses) in relation to heparin dosing? If there is, it should be defined in the manuscript.\nBlood Samples\nI assume that the systemic blood samples as a function of time will be drawn from the arterial cannula? This should be clearly indicated. This is especially important, if the authors will calculate trans-coronary concentration differences between the sinus coronarius and concomitant arterial samples. If the systemic samples will be taken from the central venous line (I assume not), such trans-coronary differences cannot be calculated.\nThe placement of the sinus coronarius catheter should be described in detail. Especially, verification of the correct placement is important. I refer to a previous publication where sinus coronaries catheter placement is verified rigorously1.\nTable 1\nThe place of the systemic samples (presumably arterial) should be indicated. The expression “3 min post cross clamp” can be understood as 3 minutes after the placement of the arterial clamp. I suggest to replace it with the expression “3 min post de-clamp”.\n\nInterim Analysis\nUsually, an interim analysis is conducted for two reasons: 1) to detect that the study hypothesis is already evident and there is no reason to continue patient recruitment; 2) to detect that the study effect is so small that it is evident that even after full recruitment there would not be statistically significant difference between the study groups. Again, there would not be any reason to continue patient recruitment. These goals are not realistic with small patient numbers of 12 (the first interim analysis) and 30 (the second interim analysis). I suggest that the primary goal of an interim analysis would not be estimation if patient recruitment should or should not be continued.\nInstead, I suggest to conduct an interim analysis after 30 patients to evaluate statistical power of the study by using the study effect (i.e. the mean difference between the intervention and control groups) and SDs at the time of analysis. If power calculation necessitates extra patients, the authors should decide a priori, how many extra patients it would be realistic to recruit. If the result of the power calculation exceeds this patient number, the RCT should be terminated and published as a negative study result. Otherwise, the patient number should be enlarged according the revised power calculation. Both interventions (doxycycline and lidocaine) should be calculated separately. It might occur, that one intervention would be terminated but the other would be continued with a large patient number. In the end, the authors would have an RCT with adequate power.\nIn theory, it is possible that there will be statistically significant difference (p<0.05) between the study groups after 30 patients. I find it highly unlikely but it is theoretically possible. In such a case, the patient recruitment should be terminated and the RCT published with a positive result.\nI admit, that also my suggestion carries a risk a premature stopping of the study. This is, however, lower than in “conventional” interim analysis, because the study power should not be weight against the intended patient number (n=20/group) but against the enlarged patient number.\nIn any case, the authors should have serious re-consideration of the present power of the study.\n\nWithdrawals\nAs the authors point out, there probably will not be many (or even a single) withdrawals or dropouts in the study. Still, the authors should be prepared to replace such patients, especially when the power calculation even with full recruitment is questionable. I suggest the following statement: “Patients whose primary outcome cannot be assessed will be replaced retaining the original allocation to the study groups.”\nPer Protocol\nI do not believe that per protocol analysis will be needed. Still, it can be included in the study protocol.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "76732", "date": "18 Jan 2021", "name": "Antonio Nenna", "expertise": [ "Reviewer Expertise Cardiac surgery." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSample size calculation should be verified after ad-interim analysis. Current sample size, considering the published details of the manuscript, seems low.\nDetails of the primary and secondary endpoints (i.e. definitions of endpoints) should be required. Fold-increase rather than crude numbers might be an option.\nConsidering baseline data, preoperative pharmacological treatments should be considered as they might lead to significant variability (i.e. statins, steroids, DMARDs,...). Inclusion/exclusion criteria should be revised accordingly to reduce variability.\nConsidering operative data, including patients with a single procedure (i.e. CABG) is generally performed to provide reliable results in this scenario. Please consider this issue; inclusion / exclusion criteria should be revised accordingly to reduce variability.\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? Not applicable", "responses": [] }, { "id": "76731", "date": "25 Jan 2021", "name": "Jong Wook Song", "expertise": [ "Reviewer Expertise Anesthesiology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis trial aims to investigate the effect of lidocaine and doxycycline on reduction of EG shedding in cardiac surgery.\nIn my opinion, assumptions for the sample size calculation seems to be too optimistic. As the authors described, and previous studies showed that syndecan-1 concentration varies wildly. The authors assumed very large treatment effect (50%). The calculation seems to based on comparison between two groups. Taken together, this trial is likely underpowered.\n\nThe investigators plan to sample coronary sinus effluent. It may provide heart-specific information. I think the investigators should describe strategy for cardioplegia administration. I'm afraid that warm blood administration before aortic de-clamping wash-out most of syndecan-1 from coronary circulation.\n\nSecondary outcome measures should be described in detail for their definition, time frame and assessment method. I think KDIGO or AKIN criteria for AKI may be more appropriate to assess kidney injury rather than delta-creatinine.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] }, { "id": "76734", "date": "02 Feb 2021", "name": "David M Baron", "expertise": [ "Reviewer Expertise Blood transfusion", "transplantation." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 submitted study protocol, the authors describe their plan to conduct an unblinded, randomized trial to explore the effects of lidocaine and doxycycline on preserving the integrity of the endothelial glycocalyx in patients undergoing surgery requiring cardiopulmonary bypass. The authors plan to investigate a total of 60 patients separated into three groups: 20 patients will receive doxycycline, 20 patients will receive lidocaine, and 20 patients will serve as control. The primary outcome is the relative difference in syndecan-1 levels, reflecting the damage to the endothelial glycocalyx.\nThe term “pilot study” seems inappropriate since the study does not aim to test feasibility of patient recruitment, data collection, or other points of the protocol in preparation for a larger study.\n\nSince the sample size calculation was based on a study including 12 pigs, interim power calculation would be rational to avoid underpowering.\n\nPlease provide details on the decisions for the specific dosages of doxycycline and lidocaine.\n\nPlease provide details on the definition of secondary outcomes and observation periods. Are you planning on using the KDIGO criteria for the categorization of acute kidney injury? Are you planning to monitor urine output as well? Since serum creatinine is a slow marker in detecting renal dysfunction, only comparing levels before induction of anesthesia with levels at admission to the ICU (see Table 1) seems insufficient. How do you define postoperative delirium?\n\nPlease provide further details on the planned statistical analyses. Which groups will be compared? Are you planning to compare all groups using, for example, ANOVA? Are you planning to compare each intervention group separately to the control group with unpaired samples t-tests?\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable", "responses": [] } ]	1	https://f1000research.com/articles/9-894
https://f1000research.com/articles/9-886/v1	04 Aug 20	{ "type": "Research Article", "title": "Decitabine treatment demethylates vast majority of high-confidence differentially methylated regions in HCT-116 colorectal cancer cells", "authors": [ "Anna Sandler", "Jonathan Anderson", "Ariane Balaram", "Yoan Ganev", "Samuel Gascoigne", "Christina Gimondo", "Brett Palmero", "Ayesha Quraishi", "Alejandro Rodriguez", "Said Omer Sadat", "William H. Conrad", "Anna Sandler", "Jonathan Anderson", "Ariane Balaram", "Yoan Ganev", "Samuel Gascoigne", "Christina Gimondo", "Brett Palmero", "Ayesha Quraishi", "Alejandro Rodriguez", "Said Omer Sadat" ], "abstract": "Background: Gene silencing by CpG island hypermethylation often plays a role in colorectal cancer (CRC) progression. Certain regions of the genome, called high confidence differentially-methylated regions (DMRs), are consistently hypermethylated across numerous patient samples. Methods: In this study, we used bioinformatics and bisulfite PCR sequencing of HCT-116 cells to investigate methylation levels at DMRs in the promoters of six genes: DKK3, EN1, MiR34b, SDC2, SPG20, and TLX1. We then investigated whether the anti-cancer drug decitabine, had a demethylating effect at these promoter regions. Results: We found that hypermethylation correlated with lack of transcriptional enhancer binding in these six regions. Importantly, we observed that for all DMRs, decitabine significantly reduced CpG methylation. Decitabine also reduced clonogenic survival, suggesting that there is a correlation between lower CpG island methylation levels and reduced cancerous properties. Conclusions: Our study provided single-nucleotide resolution and revealed hypermethylated CpG sites not shown by previous genome-wide methylation studies. In the future, we plan to perform experiments that demonstrate a causal link between promoter hypermethylation and carcinogenesis and that more accurately model treatments in CRC patients.", "keywords": [ "epigenetics", "CpG methylation", "colon cancer", "decitabine" ], "content": "Introduction\n\nColorectal cancer (CRC) is the third most common type of cancer worldwide, yet it is often caught only in its late stages1. While CRC incidence has been decreasing for individuals above 50 years of age, the incidence rates increased by 22% between 2000–2013 in individuals below 502. Chemotherapy is not effective, and patients typically survive only 44 months after the completion of treatment3. Despite this pressing reality, there is still debate about the molecular mechanisms underlying this disease. Genetic and epigenetic factors contribute to oncogenesis, but there is no consensus on a definitive molecular pathway4.\n\nCytosine methylation changes the expression of genes involved in cancer progression. DNA methylation is the transfer of a methyl group onto the C5 position of cytosine to form 5-methylcytosine5. Promoter hypermethylation has been observed to drive CRC disease progression. For instance, promoter hypermethylation of the hMLH1 gene leads to mismatch repair defects and a hypermutator phenotype in CRC6. Genetic knockout of two DNA methyltransferase genes restores expression of the tumor suppressor gene CDKN2A and slows growth of the CRC cell line HCT1167.\n\nNumerous genomic regions have been shown to be consistently hypermethylated in multiple samples of colon cancer in comparison to regular colon cells8,9. For instance, Simmer et al.10, found 2867 genomic regions consistently differentially methylated regions in CRC (DMRs). However, the genome-wide methylation techniques used by these studies lack nucleotide-level resolution at these DMRs11, and do not address if these DMRs play a mechanistic role in CRC progression.\n\nWe sought to investigate if the colorectal cancer cell line, HCT-116, can serve as a viable model for DMR hypermethylation. HCT-116 cells have been observed to be an orthotopic model for colon cancer in mice12. Furthermore, knockout of the DNA methyltransferases DNMT1 and DNMT3b has been observed to deplete >98% of total methylation and slow HCT-116 growth7, indicating global DNA methylation plays a role in HCT-116 cell survival. If HCT-116 cells are to serve as a model for DMR hypermethylation in CRC, then we expect to observe hypermethylation at established high-confidence DMRs and a reversibility of methylation with the demethylating anticancer drug, decitabine13.\n\nIn the present study, we sought to investigate the effect of the decitabine on high confidence DMRs found in the promoter regions of genes previously connected to cancer progression from 2648 available DMRs. We selected DMRs proximal to DKK3, EN1, mir34b, SDC2, SPG20, and TLX1. DKK3 is a Wnt signaling pathway inhibitor found to be hypermethylated in colon cancer cells, possibly promoting oncogenic Wnt signaling14. EN1 is a canonical gene in development15 but has recently been linked to increased cell proliferation as a non-canonical prosurvival transcription factor in cancer cells16. In addition, the EN1 promoter is found to be hypermethylated in CRC with a CpG island methylator phenotype5. mir34b is a micro-RNA that is essential for normal brain development, motile ciliogenesis and spermatogenesis17. It was found to be hypermethylated in 100 out of 101 colon cancer cell lines18. SDC2 is a transmembrane proteoglycan that mediates cytoskeletal organization and adhesion to the ECM19. SDC2 acts as a positive regulator of growth factor signals whose aberrant expression correlates with tumor size20. SDC2 promoter is hypermethylated in cancer21. SPG20 regulates cytokinesis and may alter cell division when aberrantly methylated in CRC22. TLX1 functions as a transcription factor23 and its gene promoter is frequently hypermethylated in CRC8,10. While it is overexpressed and demethylated in leukemia24 aberrant hypermethylation may also promote growth, as reviewed in 25.\n\nWe predicted that decitabine will have a negative effect on HCT-116 proliferation, and will decrease hypermethylation at these DMRs. Using the UCSC genome browser, we observed that DMR methylation inversely correlated with transcriptional activator binding across multiple cell lines. Using bisulfite PCR we observed that decitabine inhibits methylation across each DMR.\n\n\nMethods\n\nDMRs were selected from Simmer et al.10. Genomic regions from table S4 of that publication were aligned to human genome 19 (hg19) using liftOver26. UCSC genome browser was configured to visualize transcription factors and methylation status according to the following configuration: http://genome.ucsc.edu/s/williamhconrad/hg19%2Dall%2Dcell%2Dmethylation. Transcription factors were selected randomly from the track UCSC genome browser track \"Transcription Factor ChIP-seq (161 factors) from ENCODE with Factorbook Motifs\". 10 cell lines with transcription factor binding were recorded and 10 cell lines lacking transcription factor binding were recorded at each DMR. Cell lines were selected using a random number generator. Each transcription factor was recorded as a transcriptional repressor or enhancer according to UniProt27. The methylation status was then recorded for each of those cell lines. Methylation was recorded as fully methylated, mostly methylated, majority methylated, half-methylated, minority methylated, mostly unmethylated, fully unmethylated, not detected, or not tested. The cell lines were then sorted into methylated or unmethylated. The number of repressors and enhancers was recorded, and a chi squared analysis was performed to compare differences in repressor and enhancer binding to methylated or unmethylated regions using excel Data are published as underlying data28.\n\nHCT-116 cells were obtained from American type culture collection (ATCC; CCL-247) and cultured in McCoy’s 5A media (ATCC; 30-2007) in 10% fetal bovine serum (ATCC; 30-2007) and penicillin streptomycin (Thermo; 15140122). Cells were maintained in tissue culture flasks at 37 °C and 5% CO2 according to established protocols from ATCC29.\n\nTo test the effect of decitabine on the clonogenic survival of HCT-116 cells, we used protocols adapted from Franken et al.30 and Palii et al.31. Cells were plated at a density of 200 per well in a 6-well tissue culture plate. After overnight incubation, cells were treated with vehicle (anhydrous DMSO; Sigma; 276855), 0.1, 0.25, or 1 µM decitabine (Sigma; A3656). Cells were treated again after 24 hrs and then replaced with 2 ml of complete McCoy’s 5A media. After 12 days of growth, cells were fixed and stained with 10% w/v glutaraldehyde (Sigma; 340855), 0.5% w/v crystal violet (Sigma; C6158) in PBS (Sigma; 1408). After extensive rinsing in tap water, colonies were counted by eye. The average number of colonies was compared across the four conditions using a one-way ANOVA followed by Tukey’s HSD post hoc test.\n\nHCT-116 cells were cultured as described above. Exponentially growing cells were passaged to a density 20% confluency. The day following passage, cells were treated with 1 µM decitabine or DMSO. After 24 hours, treatment was repeated. After four additional days of incubation, cells were collected by trypsinization. 400,000 cells for each condition were collected and genomic DNA was isolated using the PureLink genomic DNA mini kit according to the manufacturers instructions (Thermo Fisher; K182001).\n\n400 ng of genomic DNA isolated as above was treated with 10 units of HpaII (NEB; R0171S) at 37 C for 1 hr. genomic DNA digestion was then evaluated by 1% agarose (VWR; 97062-244) gel electrophoresis. DNA was stained with 1x sybr safe (VWR; 470193-138) and imaged on a Bio-rad chemidoc imaging system.\n\n400 ng of genomic DNA isolated as above was bisulfite converted using the EZ DNA methylation kit according to the manufacturer’s instructions (zymo research; D5001). Bisulfite converted DNA was eluted in 10 µl at a concentration of approximately 40 ng / µl. Oligonucleotides for bisulfite PCR were designed using MethPrimer32. The positive strand sequence for a DMR was collected from UCSC genome browser and primers were designed for an amplicon between 150 and 400 nucleotides. Optimal annealing temperature for each primer pair were determined by testing a range of annealing temperatures between 44 and 66 °C for each primer pair against fully unmethylated and fully methylated genomic DNA (zymo; D5014). Primer pairs (Table 1), optimal annealing temperatures (Table 1), and PCR reaction conditions (Table 2), reaction master mix (Table 3) are provided in the indicated tables. Amplified DNA was purified from oligonucleotide primers and dNTPs using zymo DNA clean and concentrator-5 according to the manufacturer’s instructions (zymo; D4013). Samples were eluted in 10 µl of elution buffer and submitted for sequencing at the University of Chicago Comprehensive Cancer Center DNA sequencing and genotyping facility (Chicago, IL). Percent methylation was calculated using the relative peak height of cytosine and uracil at a given CpG site, as described previously. Peak height was quantified using Thermo Fisher Variant analysis app on the thermo fisher connect web site. Briefly, this cloud-based application processes .abi sequencing chromatogram files and returns base calls and peak height values for each peak on the chromatogram. The open source software Chromaseq can also extract identical base call and peak height values from .abi files33. Briefly, chromaseq can be installed according to their web site. The .abi sequencing files can be viewed using this software. Selecting a base call will reveal the identical peak height value presented in the chromatogram as exported in the Thermo Fisher Variant analysis app.\n\nAll statistical analyses were performed using GraphPad Prism version 7.0d. To evaluate the effect of various doses of decitabine on clonogenic survival against a no-decitabine control, a one-way ANOVA with Tukey’s post hoc was performed to control for multiple comparisons (i.e. all drug conditions against the same control). To evaluate the percent methylation of DMRs in the presence or absence of decitabine, a one-way ANOVA was performed with Bonferroni’s post hoc test to allow for multiple comparisons (i.e. between control and decitabine treated for each DMR). The specific statistical tests used are also described in the figure legends.\n\n\nResults\n\nIn mammals, cytosine methylation inversely correlates with gene expression34,35. We sought to determine if our selected DMRs might regulate gene expression. Using the UCSC genome browser36, we identified transcription factor binding data in our selected DMR regions from the encyclopedia of DNA elements (ENCODE) project37. The ENCODE project has performed 2041 transcription factor CHIP-seq experiments across 90 cell lines38,39. Transcription factor binding was observed at our DMRs (Figure 1). We hypothesized that if methylation silenced gene expression at the DMRs we selected, then we would see diminished binding of transcriptional enhancers in cell lines with methylation present at that DMR. For each of our six DMRs we observed TF binding across 20 cell lines selected by random number generator (see methods). We categorized the degree of TF binding and degree of methylation for these 10 cell lines (Underlying data for Table 428).\n\nRepresentative screen capture of the UCSC genome browser view of the DKK3 DMR. Scale depicted in track 1 (black). DKK3 gene depicted in track 2 (blue). Transcription factor binding observed in ENCODE database depicted in track 3 (grey). CpG islands depicted in track 4 (green). CpG methylation as observed by reduced representation bisulfite sequencing in HCT-116 cells depicted in track 5 (red is 100% methylated, green is 0% methylated).\n\nIndeed, we observed no transcriptional enhancers bound to methylated DMRs. Interestingly, binding of transcriptional repressors was also diminished at methylated DMRs, perhaps because methylation supplants the need for transcription factor repression. In general, transcriptional repressors and enhancers both bound more readily to cell lines with unmethylated DMRs (Table 4). The differences between repressor and enhancer binding in methylated and unmethylated DMRs was significantly different by chi-squared analysis (p<0.05). From these data, we conclude that methylation of the selected DMRs repress transcriptional enhancer binding across a broad range of cell lines.\n\nAt each DMR, transcription factor binding was evaluated across cell lines tested in the ENCODE project. No transcriptional activators were detected at methylated DMRs across cell lines. Fewer repressors were also detected at methylated DMRs. However, repressors failed to bind methylated and unmethylated DMRs with similar frequency.\n\nThe ability of a cancer cell to form a colony has been a long-standing measure for its survival in the host. Interventions that ablate clonogenicity increase patient survival40. Decitabine is known to inhibit clonogenic survival of HCT-116 cells31, and we observe inhibition of clonogenic survival at similar doses ranging from 1 µM to 100 nM (Figure 2). Furthermore, decitabine treatment is known to increase sensitivity of HCT-116 genomic DNA to the restriction enzyme, HpaII, which is inhibited by CpG methylation7. Likewise, we observe that genomic DNA collected from HCT-116 cells treated for 48 hr with 1 µM decitabine was digested by decitabine (Figure 3). From these data, we conclude that decitabine inhibits the clonogenic survival of HCT-116 cells and also inhibits DNA methylation.\n\nNumber of HCT-116 colonies detected by crystal violet staining 14 days after two 24-hour treatments of the indicated doses of decitabine. p<0.05 ANOVA, Tukey's post-hoc.\n\nHCT-116 cells were treated with 1 µM decitabine or DMSO for 48 hrs. Genomic DNA was extracted and treated with the methylation-sensitive enzyme, HpaII as indicated. 400 ng of gDNA was separated by gel electrophoresis. Ladder indicated to the left of the gel.\n\nAfter observing global demethylation by 1 µM decitabine (Figure 3), we next sought to determine the degree of CpG methylation at DMRs in HCT-116 cells, and if those methylation sites were inhibited by decitabine. Using bisulfite PCR, we detected conversion of unmethylated cytosine to uracil (Figure 4a). Importantly, we both identified methylated CpG sites previously detected by reduced-representation bisulfite sequencing as part of the ENCODE project, and we also identified novel CpG sites in the region, adding resolution to the methylation status of these select DMRs (Figure 4b).\n\n(a) Representative chromatograms of bisulfite PCR / sanger sequencing of the DKK3 DMR. Percent methylation quantified by peak height of C relative to C + T. (b) Mapping of bisulfite PCR results onto human genome 19. Scale depicted in track 1 (black). DKK3 gene depicted in track 2 (blue). CpG islands depicted in track 3 (green). CpG methylation as observed by reduced representation bisulfite sequencing in HCT-116 cells depicted in track 4 (red is 100% methylated, green is 0% methylated). Bisulfite PCR depicted in track 5 (DMSO) and track 6 (decitabine treated). The two grey bars show region sequenced. The red-green spectrum depict degree of methylation. (c) Quantification of percent methylation of sites depicted in (b). Percent methylation for each DMR as in (c). p<0.05 ANOVA with Bonferroni’s post-hoc test. Columns and bars are mean and SEM, respectively.\n\nWe quantified the degree of CpG methylation at each site detected by bisulfite PCR in the presence or absence of decitabine. We observed statistically significant reductions in CpG methylation at all tested DMRs. From these data we can conclude that all colon cancer DMRs tested are hypermethylated in HCT-116 cells, that bisulfite PCR offers increased resolution at DMRs over HM450 array or RRBS, and that methylation at DMRs is reversable by 1 µM decitabine treatment.\n\n\nDiscussion\n\nFrom our results, we conclude that HCT-116 cells can serve as a model for investigating the role of high confidence DMRs in colon cancer. As observed previously, we also observed HCT-116 cells to be sensitive to the demethylating agent decitabine7,31. Decitabine inhibited clonogenicity (Figure 2) and demethylated genomic DNA (Figure 3). By bisulfite PCR, we observed DMRs previously identified in patient colon cancer cells to be consistently hypermethylated in HCT-116 cells. Furthermore, we achieved single-nucleotide resolution of CpG methylation at these DMRs. We were able to confirm previously identified CpG sites as well as identify new ones (Figure 4).\n\nThe HCT-116 model of DMR methylation has potential to shed light on the role of DMRs in colon cancer pathogenesis. Moving forward, it will be important to determine if (a) DMRs reduce nearby gene expression, (b) if DMR demethylation increases gene expression, and (c) if restoring expression of methylation-silenced genes affects HCT-116 growth or survival. In future, we plan to increase resolution at DMRs relevant to HCT-116 cell growth by sequencing across the entire DMR and by testing additional DMRs. The most updated data can be viewed using the UCSC genome browser public session found here: https://bit.ly/UCSC-DMR-methylation. The original data presented in this manuscript will be maintained as described in the data availability section28,41.\n\nFuture work is needed to address the question if specific gene products suppressed by hypermethylation play a role in tumor growth. Nine frequently hypermethylated genes have been observed to slow cancer cell growth when heterologously expressed42. Two additional frequently hypermethylated genes have been observed to inhibit cancer cell colony formation when heterologously expressed43. Moving forward, we seek to heterologously express gene products suppressed by hypermethylation to test if such expression affects tumor cell growth and colony formation.\n\n\nData availability\n\nZenodo: williamhconrad/HCT116-DMR-bisulfite-PCR: bisulfite PCR repository under CC0 license. http://doi.org/10.5281/zenodo.394843928\n\nThis project contains the following underlying data:\n\n“Underlying data for Figure 2 - clonogenic survival.xlsx” (a spreadsheet containing the clonogenic survival data depicted in figure 2)\n\n“Underlying data for Figure 4b - UCSC methylation tracks for DMSO.bed” (a spreadsheet (tab-delimited bed format) containing the percent methylation data for DMSO treated cells presented in figure 4b)\n\n“Underlying data for Figure 4b - UCSC methylation tracks for decitabine.bed” (a spreadsheet (tab-delimited bed format) containing the percent methylation data for decitabine treated cells presented in figure 4b)\n\n“Underlying data for Figure 4c and d raw methylation quantification.xlsx” (a spreadsheet containing the raw methylation data data depicted in figures 4c and d)\n\n“Underlying data for table 4 DMR TF dataset-FINAL.xlsx” (a spreadsheet containing the transcription factor binding data for DMRs evaluated in table 4)\n\nfig 3 raw hpaii digest image 300 dpi.tif (Raw gel image for Figure 3)\n\nZenodo: williamhconrad/HCT116-decitabine-hub: Decitabine Hub repository under CC0 license. http://doi.org/10.5281/zenodo.394675341.\n\nThis project contains the following underlying data that are used to build the UCSC genome browser public hub found at https://bit.ly/UCSC-DMR-methylation:\n\n“description.html” (An html file that describes the Tracks in this repository. This html file is used by UCSC genome browser to build a description for the public hub)\n\n“description.fld” (A folder with formatting for the file “description.html”)\n\n“genomes.txt” (A file used by UCSC genome browser to select the correct genome to annotate the methylation data)\n\n“hub.txt” (A file used by UCSC genome browser to find the DNA methylation tracks )\n\n“ BMB322L-pctMethyl-DMSO-20200602.bb”, “ BMB322L-pctMethyl-DMSO-20200602.bed”, and “ BMB322L-pctMethyl-DMSO-20200602.bed” (identical spreadsheets in three formats containing the percent methylation data for DMSO treated cells presented in figure 4b for use by UCSC genome browser. The “.bb” and “bigBed” files are in bigbed format, the “.bed” file is in bed format.)\n\n“ BMB322L-pctMethyl-Decitabine-20200602.bb”, “ BMB322L-pctMethyl- Decitabine -20200602.bed”, and “ BMB322L-pctMethyl- Decitabine -20200602.bed” (identical spreadsheets in three formats containing the percent methylation data for Decitabine treated cells presented in figure 4b for use by UCSC genome browser. The “.bb” and “bigBed” files are in bigbed format, the “.bed” file is in bed format.)\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\nDe Rosa M, Pace U, Rega D, et al.: Genetics, diagnosis and management of colorectal cancer (Review). Oncol Rep. 2015; 34(3): 1087–96. PubMed Abstract \| Publisher Full Text \| Free Full Text\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): 2893–917. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPaz MF, Wei S, Cigudosa JC, et al.: Genetic unmasking of epigenetically silenced tumor suppressor genes in colon cancer cells deficient in DNA methyltransferases. Hum Mol Genet. 2003; 12(17): 2209–19. PubMed Abstract \| Publisher Full Text" }	[ { "id": "69566", "date": "27 Aug 2020", "name": "Keith D. Robertson", "expertise": [ "Reviewer Expertise epigenetics", "DNA methylation", "cancer" ], "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\nReview comments: This study investigates DNA methylation levels of DMRs in a single-nucleotide resolution using bisulfite sequencing. The selected DMRs are at the promoter regions of six genes based on published data from others. HCT116, a commonly used cell line in CRC cancer served as an acceptable model in this study. Decitabine (5-aza-2'-deoxycytidine) treatment in HCT116 significantly reduced CpG methylation at the DMRs, as well as clonogenic survival after a 5-day treatment, all consistent with published data. Overall, while the study is thoughtfully put together, the scope is quite limited, but it can make a certain level of contribution to CRC research. The number of studies using HCT116 and decitabine is very large, and the authors would benefit from additional literature searches to put their data in to the context of what has been done in this area, which is extensive. Thus a key question with regard to publish/not publish, is whether the manuscript adds to what is already known. It is possible that for these loci, the data may be novel, but then again many studies have used HCT116 and decitabine with various omics methods (e.g. 450k array) so it would be worth checking if the sites examined here overlap with those in omics analyses to help determine novelty of what is shown here.\nThe manuscript is well written. However, there are a number of issues with the methods and analysis that need to be clarified/addressed or repeated. Adding a piece of extra bioinformatic data and RT-PCR results are also necessary to make clarify the study conclusions. These issues, and those mentioned above, prevent me to endorse its acceptance at the present stage. Below are more specific comments by section.\n\nPoints to address: Replacing the most recently updated literature about CRC epidemiology, biology and epigenetics, and more accuracy of descriptions of what this literature has shown to improve the relevance of the study. As mentioned above there have probably been 100’s of papers looking at methylation in CRC and using HCT116 cells as a model. Use of TCGA CRC data, which may or may not encompass the CpGs examined (they used the 450k array) is also strongly recommended.\n\nThe in silico analysis of transcription factor binding in relation to DNA methylation is clever, however the only really way to test this effect formally is to do experiments like EMSA with methylated/unmethylated problems and ChIP on methylated or unmethylated cells. So the outcome of the in silico analysis should be highly qualified.\nThe authors selected six DMRs proximal to DKK3, EN1, mir34b, SDC2, SPG20, and TLX1 genes from 2648 available DMRs. As all these DMRs were identified in 24 tumors and matched normal colon samples in a previous publication, due to the heterogeneity of CRC, it is hard to conclude that the methylation status behaves similarly in CRC cancer cell lines. There is a publicly available database (CCLE-https://portals.broadinstitute.org/ccle) in which DNA methylation data (RRBS) and RNA-seq results can be extracted for certain cancer cell lines including HCT116 and other 59 CRC cell lines. Integrating this piece of data will help to validate choice of HCT116 cells as a viable model for primary CRC and why these 6 DMRs were picked up for validation in this study.\nRationale for choosing these DMRs needs to be improved. RT-PCR for gene expression is necessary to show the inverse relationship between DNA methylation and gene expression. It will also support the results found in the transcriptional enhancer binding analysis.\nTranscriptional enhancer binding analysis: DMR Methylation was arbitrarily recorded and put into different methylation groups. Can cut off values be provided to support regrouping? Clonogenic survival assay: It is better to seed cells after drug treatments to remove the confounding effect caused by the toxicity of decitabine. It is hard to explain why there is no colony formed even using 0.25µM of decitabine. The clonogenic survival assay should be redone to remove the confounding effect. But again, this assay has already been done by others. What would add true novelty here would be to CRISPR target a single hypermethylated gene, then do a clonogenic assay.\nGlobal demethylation analysis: Recommend to use MspI-HpaII pair rather than HpaII itself. MspI is an isoschizomer of HpaII which cleaves both unmethylated and methylated HpaII sites, but HpaII is unable to cleave the site when the inner CG dyad is fully- or hemi- methylated. Similar digestion patterns will be observed when unmethylated DNA was treated with MspI and HpaII, while different patterns will show up for methylated DNA.\nBisulfite PCR and sequencing: In most publications, bisulfite PCR and sequencing clones is preferred. The authors should detail the accuracy of their method using the relative peak height of cytosine and uracil at a given CpG site to calculate percent methylation. It seems like standards would be needed for this method to be quantitative.\nTable 2 and 3 can either be supplementary tables or be described in the context of the methods section. It is not necessary to list as tables in the main section.\nResults P5: The author stated that “In mammals, cytosine methylation inversely correlates with gene expression”. It seems true mostly in cases of DNA methylation in the promoter regions, but not in gene body. Please qualify.\nP5: Methylation inversely correlates with transcriptional enhancer binding: Is there specific supporting data for HCT116? Table 5: Chi square calculation is not correct (the expected values) for table 4 in the underlying data.\nFigure 2: There is no colony formation when HCT 116 is treated by 0.25µM and 1 µM decitabine. It might be caused by non-specific drug toxicity. Repeating this experiment by plating cells after drug treatments is strongly recommended.\nFigure 3: What causes difference of bands in column 1 and 3? It is better to add MspI digestion beside HpaII.\nFigure 4b: Why are the length of the sequenced regions different for DMSO and decitabine? There are fewer CpGs sequenced in the decitabine treatment group than in the DMSO group. Figure 4c and 4d: As different numbers of CpGs are included for the 2 treatment groups (DMSO and decitabine), comparison of the methylation percentage between groups is biased. Because this is a main result in this study, the authors need to deal with this problem.\nDiscussion first sentence on P.6 in this section (“From our results, we conclude that HCT-116 cells can serve as a model …” ): the conclusion overreaches based on the data collected. Six DMRs cannot represent the whole picture of DNA methylation in primary CRC.\nEven though the authors planned to test gene expression near the selected DMRs in the future, it is recommended to add the gene expression data in the current study to make a better story.\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?\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": [] }, { "id": "75476", "date": "11 Dec 2020", "name": "Matjaz Rokavec", "expertise": [ "Reviewer Expertise Cancer research" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study, Sandler et al. investigated methylation status of six genes (DKK3, EN1, MiR34b, SDC2, SPG20, and TLX1) in colorectal cancer. By using the HCT116 CRC cell line as a model for CRC, the authors show that all six analzed genes are methylated. Using bioinformatics approaches, they show that methylation inhibited transcriptional enhancer binding. Treatment of HCT116 cells with the demethylation compound Decitabine (5-Aza-2'-desoxycytidin) decreased the methylation of all genes. Finally, Decitabine treatment inhibited the clonogenic survival of HCT116 cells.\nThe six genes were selected based on a previous genome-wide study that identified 2648 differentially methylated regions in CRC. However, Sandler et al. did not describe what was the criteria to select the six analyzed genes.\nBy performing bisulfite sequencing they provide methylation status of the six genes at single nucleotide resolution. However, Cancer Cell Line Encyclopedia (CCLE) provides RRBS genome-wide methylation analyzes of 59 CRC cell lines. Therefore, the methylation status of the six genes can be also extracted from CCLE data.\nThe bioinformatics analyzes suggest that methylation inhibits transcriptional enhancer binding to the six genes. However, these findings needs to be experimentally validated before drawing any conclusions.\nIt has been shown in numerous studies that Decitabine inhibits the viability and clonogenic survival of various cancer cell lines, including HCT116. Therefore, this part of the study does not provide any new findings.\nAltogether, I believe that the novelty and the study design are not sufficient for indexing.\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": [] } ]	1	https://f1000research.com/articles/9-886
https://f1000research.com/articles/9-878/v1	03 Aug 20	{ "type": "Systematic Review", "title": "Re-thinking antimicrobial resistance transmission dynamics: a meta-analysis of cross-sectional studies at referral hospitals in Uganda", "authors": [ "Gerald Mboowa", "Ivan Sserwadda", "Dickson Aruhomukama", "Gerald Mboowa", "Ivan Sserwadda" ], "abstract": "Background: Antimicrobial resistance threatens the achievements of modern medicine as well as the sustainability of effective global public health responses to the threat posed by infectious diseases. Extended-spectrum β-lactamase production in bacteria provides the main mechanism of resistance in gram-negative bacteria, particularly those belonging to the Enterobacteriaceae family as well as gram-positive bacteria. This study hence aimed at providing insights into the potential role of in-patients, their immediate hospital environments, out-patients, and their communities in the transmission of antimicrobial resistance via identifying gram-negative and gram-positive bacteria commonly isolated in samples collected from each of these patients/sites as well as their antimicrobial susceptibility profiles using extended-spectrum β-lactamase production in the same as the basis. Methods: Our study reviewed four cross-sectional studies conducted at national and regional referral hospitals in Uganda. Data on bacterial aetiology and antimicrobial susceptibility testing retrieved from the studies was imported into Microsoft Excel, cleaned, sand then exported to IBM SPSS statistics (version 16) for statistical analysis. The databases used were PubMed and Embase. Results: We report that; Escherichia coli and Klebsiella pneumoniae are the most prevalent Enterobacteriaceae species in the samples that were collected in the studies reviewed; these species account for the highest proportions of extended-spectrum β-lactamase producers; Staphylococcus aureus is the most prevalent of the gram-positive bacteria isolated from the same samples, and accounts for the highest proportions of extended-spectrum β-lactamase producers in the gram-positive bacteria isolated, and similar Enterobacteriaceae species and gram-positive bacteria, are predominant in samples from in-patients, their immediate hospital environments, and out-patients. Conclusion: The insights provided indicate antimicrobial resistance transmission dynamics be re-thought and more comprehensive studies aimed at investigating the same be done to ascertain the source and transmission routes of antimicrobial-resistant bacteria in clinical settings.", "keywords": [ "Antimicrobial resistance", "Transmission dynamics", "Extended-spectrum β-lactamases", "Uganda" ], "content": "Introduction\n\nAntimicrobial resistance not only threatens the achievements of modern medicine but also the sustainability of effective global public health responses to the threat posed by infectious diseases1,2. Extended-spectrum β-lactamase production in bacteria confers antimicrobial resistance and provides the main mechanism of resistance in gram-negative bacteria, particularly those belonging to the Enterobacteriaceae family as well as gram-positive bacteria3,4. Extended-spectrum β-lactamase producing bacteria exhibit resistance to all penicillin-like antibiotics except cephamycins and carbapenems; resistance in these bacteria frequently spans to other classes of antibiotics, including the quinolones and aminoglycosides5–7. Resistance to β-lactam antibiotics is mostly plasmid-borne and can be disseminated among bacterial species via horizontal gene transfer; this has consequently led to clonal distribution within and between clinical settings, communities as well as across local and international boarders via patient mobility7,8. Combating antimicrobial resistance requires continuous and timely monitoring, surveillance, and assessment systems as well as mechanisms to detect extended-spectrum β-lactamase producing bacteria. In Uganda, the acquisition and transmission dynamics of extended-spectrum β-lactamase producing bacteria at referral hospitals and community settings have not been elucidated. The aim of this study was therefore to provide insights into the potential role of in-patients, their immediate hospital environments, out-patients, and their communities in the transmission of extended-spectrum β-lactamase producing bacteria via identifying gram-negative and gram-positive bacteria commonly isolated in samples collected from each of these patients/sites as well as their antimicrobial susceptibility profiles using extended-spectrum β-lactamase production in the same as the basis.\n\n\nMethods\n\nWe conducted a systematic review of cross-studies on bacterial aetiology and antimicrobial susceptibility profiles conducted at national and regional referral hospitals in Uganda.\n\nThe application of the inclusion and exclusion criteria was undertaken independently by the reviewers (GM, IS, and DA) and any difference in opinion was resolved by discussion among the reviewers. Also, the inclusion and exclusion criteria were the same for both the systematic review as well as the meta-analysis.\n\nIncluded studies were as follows:\n\nStudies whose designs were cross-sectional.\n\nStudies that were conducted in Ugandan national and regional referral hospitals.\n\nStudies that reported bacterial aetiology and antimicrobial susceptibility profiles.\n\nStudies with the requirements above that had been published within the last ten years.\n\nExcluded studies were as follows:\n\nStudies that had been published as reviews\n\nStudies in which data had been reported only as abstracts.\n\nStudies whose designs were not cross-sectional (i.e. retrospective and prospective)\n\nSearch strategy. The Pubmed and Embase electronic databases were searched for eligible studies. We used the search string [“Antimicrobial resistance”] AND [“Referral Hospitals”] AND [“Uganda”]. We also sought additional studies through screening the references of the selected studies.\n\nData extraction was done by GM, IS and DA. Data were entered in prespecified spreadsheet in Microsoft Excel (2019 version). The extracted data elements consisted of (1) bacterial aetiology and (2) antimicrobial susceptibility profiles.\n\nRisk of bias. For the bias risk analysis, an appraisal tool for cross-sectional studies, AXIS was used9. In addition, quality assessment of the included studies referred to a modified method composed of the design of the studies, participant, sites and settings selection methodology and laboratory tests that included; determination of bacterial aetiology, antimicrobial susceptibility testing and screening for the production of Extended-spectrum β-lactamases (extended data10).\n\nStatistical analysis. The main outcomes of interest in the data analysis were: 1) the proportion of gram-positive and negative bacteria isolated from the samples collected in the studies; and 2) the proportion of the gram-positive and negative bacteria isolated that were producers of Extended-spectrum β-lactamases.\n\nFor all the selected studies, data on bacterial aetiology and antimicrobial susceptibility testing was extracted using Microsoft Excel (2019 version). Following data extraction, several tables containing dichotomous data were generated for the relative and cumulative calculation of the frequencies of the outcomes of interest. For all of the meta-analysis procedures, IBM SPSS Statistics (version 16) was used. In SPSS, forest and funnel plots and charts were generated. The proportions of either the gram- positive and negative bacteria was determined by the number of the same divided by the total number bacteria isolated in the studies. A similar approach was used to determine the proportions of gram-positive and negative bacteria isolated that were producers of Extended-spectrum β-lactamases. The I2 test was used to access the existence of heterogenicity of between the studies (I2 = 75–100%, p<0.05)11,12. Due to the nature of the studies, heterogenicity was expected; we therefore chose to use the random effects model for the meta-analysis as proposed by DerSimonian-Laird13. We also performed a sensitivity analysis to test for the effect of the individual influence of each study on the overall estimate, and a subgroup analysis was also performed to reduce the existence of heterogenicity. In addition, we evaluated the existence of publication bias by visual inspection of Begg’s funnel plots as well as by Egger’s test calculation14,15.\n\n\nResults\n\nPrimarily, during the search for studies in the digital databases as well as additional records from other sources, 52 reference studies were found. After the application of the inclusion and exclusion criteria, we refined the results, and 4 eligible studies constituted the present meta-analysis16–19. The flowchart of this selection is shown in the PRISMA diagram is shown in Figure 1.\n\nOf the 4 studies included in the meta-analysis, 2/4 (i.e. Study 1 (Seni et al., 2013) and Study 2 (Masifa et al., 2018)) collected samples from in-patients; the samples collected in the two studies included wound and pus swabs, these samples had only been obtained from patients with wound sepsis based on evidence of post-operative wound infection during the respective study periods (i.e. September 2011 to April 2012 and June to October 2015 respectively) from the General Surgery, Obstetrics and Gynecology, Orthopedic and General Surgery, Obstetrics and Gynecology wards respectively16,17; 1/4 (i.e. Study 3 – Kateregga et al., 2015) collected samples from both in-patients and out-patients, the samples collected included urine, swabs (i.e. oral, high vaginal, and wound), blood, and cerebrospinal fluid; the samples had been collected by purposive sampling from patients attending different hospital wards; these wards included Surgical wards (i.e. General Surgical and Surgical Out-patient Department), Medical wards (i.e. General, Medical Out-patient Department, Medical assessment Center, Uganda Cancer Institute-Liquid Tumor Cancer, and Uganda Heart Institute), Pediatric wards, General wards (i.e. Out-patient Department, Medical Out-patient Department, and the Sexually Transmitted Diseases Department); the samples were collected from January to April 201418; 1/4 (Study 4 – Sserwadda et al., 2018) collected samples from the hospital environment, specifically, from medical equipment (i.e. scissors, infusion stands), patients’ beds and work surfaces (i.e. tables, sink taps and light switches); the samples had been collected by purposive sampling from the different sites, consideration was given to sites with constant hand contact, and the samples were collected from June to December 201519.\n\nStudies 1, 2, 3, and 4 reported Enterobacteriaceae making up 47.7%, 49%, 46.9%, and 25.6% out of the total isolates obtained from these respective studies (Table 1). Escherichia coli was found to be the most predominant of the Enterobacteriaceae isolates obtained from studies 1, 2, and 3 accounting for 49.7%, 43.1%, and 53.9% respectively; second to Escherichia coli was Klebsiella pneumoniae, this accounted for 12.8%, 29.4%, and 28.7% of the Enterobacteriaceae isolates in studies 1, 2, and 3 respectively. In a similar regard, Klebsiella pneumoniae was found to be the most predominant of the Enterobacteriaceae isolates in study 4 representing 46.7%; second to Klebsiella pneumoniae was Proteus vulgaris, this represented 33.3% of the Enterobacteriaceae isolates (Table 1).\n\nExtended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae were reported in all the four studies, these represented 81.4%, 76.5%, 62% and 20% for studies 1, 2, 3 and 4 respectively. In studies 1 and 2, Escherichia coli was found to be the main extended-spectrum β-lactamase producer accounting for 48.3% and 41% respectively while Klebsiella pneumonia was the main extended-spectrum β-lactamase producer for studies 3 and 4 accounting for 72.7% and 100% respectively (Table 1).\n\nKey: Study 1 – Seni et al., 2013; Study 2 – Masifa et al., 2018; Study 3 – Kateregga et al., 2015; Study 4 – Sserwadda et al., 2018; ESBL - Extended-spectrum β-lactamase\n\nStaphylococcus aureus was the most predominant gram-positive isolate obtained from studies 1, 2 and 4 accounting for 67.4%, 93.2% and 75.4% respectively (Table 2). Study 3 did not isolate any gram-positive bacteria as it only considered gram-negative bacteria (Table 2). Proportions of extended-spectrum β-lactamase producing Staphylococcus aureus were 67.4%, 75% and 70.5% for studies 1, 2 and 4 respectively. Study 3 did not isolate any gram-positive bacteria and therefore no extended-spectrum β-lactamase production was reported (Table 2).\n\nKey: Study 1 – Seni et al., 2013; Study 2 – Masifa et al., 2018; Study 3 – Kateregga et al., 2015; Study 4 – Sserwadda et al., 2018; NA- No gram-positive bacteria isolated; ESBL - Extended-spectrum β-lactamase.\n\n\nDiscussion\n\nThis study reviewed four cross-sectional studies on bacterial aetiology and antimicrobial susceptibility profiles conducted at referral hospitals in Uganda with the aim of providing insights into the potential role of in-patients, their immediate hospital environments, out-patients, and their communities in the transmission of antimicrobial resistance via identifying gram-negative and gram-positive bacteria commonly isolated in samples collected from each of these patients/sites as well as their antimicrobial susceptibility profiles using extended-spectrum βlactamase production in the same as the basis.\n\nThis study achieved the objectives for which it was conducted; the major findings were that; (i) Escherichia coli and Klebsiella pneumoniae are the most prevalent Enterobacteriaceae species in the samples that had been collected in the studies reviewed; (ii) these species (i.e. Escherichia coli and Klebsiella pneumoniae) account for the highest proportions of extended-spectrum β-lactamase producers; (iii) Staphylococcus aureus is the most prevalent of the gram-positive bacteria isolated from the same samples, and accounts for the highest proportions of extended-spectrum β-lactamase producers in the gram-positive bacteria isolated, and (iv) similar Enterobacteriaceae species and gram-positive bacteria are predominant in samples from in-patients, their immediate hospital environments, and out-patients.\n\nThe findings of this study with regards to Escherichia coli and Klebsiella pneumoniae being the most prevalent Enterobacteriaceae species in the samples are consistent with those of similar studies; these studies have not only highlighted these bacterial species to be the most prevalent in samples from in-patients20–24, but have also indicated them to be the most prevalent in samples from out-patients as well in those from the hospital environment7,25–27. The findings of this study with regards to the isolation of similar Enterobacteriaceae species from in-patient samples, those from their immediate hospital environments, and out-patients could be suggestive of the possibility of complex transmission dynamics of the same.\n\nIn a similar light, despite the fact that these remain the most prevalent Enterobacteriaceae species, this study as well the studies reviewed did not ascertain the source of these; however, speculate that these could be from both nosocomial and community sources.\n\nIn addition, the findings of this study with regards to these Enterobacteriaceae species being the main extended-spectrum β-lactamase producers of the gram-negative isolates are consistent with those of similar studies20,22.\n\nThe prevalence of these species in samples collected from both the community and hospital settings continues to increase. The source of ESBL-PE in samples from the community could be as a consequence of the introduction by individuals that had visited health-care settings or had interacted with health-care workers in the same settings, this has also been suggested in other studies28,29. Some studies have however suggested that population exposures to antibiotics could offer an explanation to the observation25,30, while some have demonstrated carriage of the same bacteria in individuals with no history of having taken antibiotics31 and have highlighted food products from animals as potential sources of the bacteria32.\n\nAccording to the 2017 World Health Organization list of priority pathogens, these Enterobacteriaceae species have been identified to belong to the priority one or critical category, and particularly when these are producers of extended-spectrum β-lactamases. Extended-spectrum β-lactamases confer resistance to all penicillin derivative antibiotics as well as cephalosporins, except cephamycins and carbapenems7,33; despite the fact that this study and the studies reviewed did not ascertain which extended-spectrum β-lactamase encoding genes were mediating the observed β-lactam resistance and how these are potentially disseminated in the isolates, our study speculates that the extended-spectrum β-lactamase encoding genes could have been either or a combination of the blaCTX-M, blaSHV and blaTEM, and that horizontal gene transfer could be a plausible mechanism by which the dissemination of these genes could be occurring. This is because, not only have most extended-spectrum β-lactamase encoding genes described so far been derivatives of the blaCTX-M, blaSHV and blaTEM, but these have also been reported to be the most prevalent in isolates just like those in the studies reviewed; these genes have also been documented to be mostly encoded by conjugative plasmids; these are mobile genetic elements (MGEs)7,8,33,34.\n\nBesides extended-spectrum β-lactamase encoding genes, these MGEs have also been documented to carry genes that confer resistance to other antibiotic classes that include fluoroquinolones, aminoglycosides, and trimethoprim/sulfamethoxazole; these have also been documented to contribute to the selection and persistence of multidrug-resistant extended-spectrum β-lactamase producing strains in samples from in-patients, their immediate hospital environments, and out-patients; this could explain the resistance to other antibiotics observed in the studies reviewed5,7,8,35,36.\n\nThe findings of our study with regards to Staphylococcus aureus being the most prevalent of the gram-positive bacteria isolated from the samples collected in the studies, and accounting for the highest proportions of extended-spectrum β-lactamase producers in the gram-positive bacteria species from in-patient samples, those from their immediate hospital environments, and out-patients are consistent with those of similar studies; these studies have not only highlighted these bacterial species to be the most prevalent in samples from in-patients, but have also indicated them to be the most prevalent in samples from out-patients, as well in those from the hospital environments37–39. In a similar light, despite the fact that these remain the most prevalent gram-positive bacteria species, our study as well as the studies reviewed did not ascertain the source of these, however, similar to the case of the Enterobacteriaceae species, this study speculates that these could be from both nosocomial and community sources.\n\nAccording to the 2017 World Health Organization list of priority pathogens, Staphylococcus aureus, has been identified to belong to the priority two or high category, and particularly when these are producers of extended-spectrum β-lactamases or which exhibit methicillin-resistance (MRSA). Despite the fact that MRSA has long been a pathogen of nosocomial origin, in recent times, it has emerged as a problematic pathogen in community settings40,41. These same studies have also indicated the possibility of community-associated MRSA being replaced by nosocomial-associated MRSA.\n\n\nConclusion\n\nThe results of our study indicate that similar extended–spectrum β-lactamase producing Enterobacteriaceae species and gram-positive bacteria are predominant in samples from in-patients, their immediate hospital environments, and out-patients. This study offers insights into the potential role of in-patients, their immediate hospital environments, out-patients, and their communities in the transmission of antimicrobial resistance. In addition, the results of our study, underscores the need for continuous monitoring and surveillance of antimicrobial resistance in clinical and community settings, together with efforts to promote the rational and judicious use of antibiotics. Lastly, with the insights provided here, antimicrobial resistance transmission dynamics should be re-thought and more comprehensive studies aimed at investigating the same should be done to ascertain the source and transmission routes of antimicrobial resistant bacteria in community and clinical settings.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: S1 File.docx. https://doi.org/10.6084/m9.figshare.12563153.v110\n\nThis project contains the following extended data:\n\n- S1 File.docx (Summary of included study characteristics)\n\nFigshare: PRISMA checklist for ‘Re-thinking antimicrobial resistance transmission dynamics: a meta-analysis of cross-sectional studies at referral hospitals in Uganda’ https://doi.org/10.6084/m9.figshare.12563318.v142\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nUtt E, Wells C: The global response to the threat of antimicrobial resistance and the important role of vaccines. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoyo SJ, Aboud S, Kasubi M, et al.: Antimicrobial resistance among producers and non-producers of extended spectrum beta-lactamases in urinary isolates at a tertiary Hospital in Tanzania. BMC Res Notes. 2010; 3(1): 348. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaina D, Revathi G, Kariuki S, et al.: Genotypes and cephalosporin susceptibility in extended-spectrum beta-lactamase producing enterobacteriaceae in the community. J Infect Dev Ctries. 2012; 6(06): 470–7. PubMed Abstract \| Publisher Full Text\n\nMpogoro FJ, Mshana SE, Mirambo MM, et al.: Incidence and predictors of surgical site infections following caesarean sections at Bugando Medical Centre, Mwanza, Tanzania. Antimicrob Resist Infect Control. 2014; 3(1): 25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChahoud J, Kanafani Z, Kanj SS: Surgical site infections following spine surgery: eliminating the controversies in the diagnosis. Front Med (Lausanne). 2014; 1: 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Lissovoy G, Fraeman K, Hutchins V, et al.: Surgical site infection: incidence and impact on hospital utilization and treatment costs. Am J Infect Control. 2009; 37(5): 387–97. PubMed Abstract \| Publisher Full Text\n\nNajjuka CF, Kateete DP, Kajumbula HM, et al.: Antimicrobial susceptibility profiles of Escherichia coli and Klebsiella pneumoniae isolated from outpatients in urban and rural districts of Uganda. BMC Res Notes. 2016; 9(1): 235. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMuhammad UK, Isa MA, Aliyu ZM: Distribution of potential nosocomial pathogens isolated from environments of four selected hospital in Sokoto, North Western Nigeria. J Microbiol Biotechnol Res. 2013; 3(1): 139–43.Reference Source\n\nEkrami A, Kayedani A, Jahangir M, et al.: Isolation of common aerobic bacterial pathogens from the environment of seven hospitals, Ahvaz, Iran. 2011. Reference Source\n\nNaseer U, Haldorsen B, Simonsen GS, et al.: Sporadic occurrence of CMY-2-producing multidrug-resistant Escherichia coli of ST-complexes 38 and 448, and ST131 in Norway. Clin Microbiol Infect. 2010; 16(2): 171–8. PubMed Abstract \| Publisher Full Text\n\nYoung BE, Lye DC, Krishnan P, et al.: A prospective observational study of the prevalence and risk factors for colonization by antibiotic resistant bacteria in patients at admission to hospital in Singapore. BMC Infect Dis. 2014; 14(1): 298. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWoerther PL, Angebault C, Jacquier H, et al.: Characterization of fecal extended-spectrum-β-lactamase-producing Escherichia coli in a remote community during a long time period. Antimicrob Agents Chemother. 2013; 57(10): 5060–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBailey JK, Pinyon JL, Anantham S, et al.: Commensal Escherichia coli of healthy humans: a reservoir for antibiotic-resistance determinants. J Med Microbiol. 2010; 59(Pt 11): 1331–9. PubMed Abstract \| Publisher Full Text\n\nOverdevest I, Willemsen I, Rijnsburger M, et al.: Extended-spectrum β-lactamase genes of Escherichia coli in chicken meat and humans, The Netherlands. Emerg Infect Dis. 2011; 17(7): 1216–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAruhomukama D: Phenotypic Assays for Detection of Extended Spectrum Β-Lactamases and Carbapenemases: A Laboratory Guide for Microbiologists. 2018. [cited 2018 Sep 24]. Reference Source\n\nDoi Y, Adams-Haduch JM, Peleg AY, et al.: The role of horizontal gene transfer in the dissemination of extended-spectrum beta-lactamase–producing Escherichia coli and Klebsiella pneumoniae isolates in an endemic setting. Diagn Microbiol Infect Dis. 2012; 74(1): 34–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoura A, Henriques I, Ribeiro R, et al.: Prevalence and characterization of integrons from bacteria isolated from a slaughterhouse wastewater treatment plant. J Antimicrob Chemother. 2007; 60(6): 1243–50. PubMed Abstract \| Publisher Full Text\n\nBush K: Alarming β-lactamase-mediated resistance in multidrug-resistant Enterobacteriaceae. Curr Opin Microbiol. 2010; 13(5): 558–64. PubMed Abstract \| Publisher Full Text\n\nSalgado CD, Farr BM, Calfee DP: Community-acquired methicillin-resistant Staphylococcus aureus: a meta-analysis of prevalence and risk factors. Clin Infect Dis. 2003; 36(2): 131–9. PubMed Abstract \| Publisher Full Text\n\nDavid MZ, Daum RS: Community-associated methicillin-resistant Staphylococcus aureus: epidemiology and clinical consequences of an emerging epidemic. Clin Microbiol Rev. 2010; 23(3): 616–87. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHiramatsu K, Cui L, Kuroda M, et al.: The emergence and evolution of methicillin-resistant Staphylococcus aureus. Trends Microbiol. 2001; 9(10): 486–93. PubMed Abstract \| Publisher Full Text\n\nPopovich KJ, Weinstein RA, Hota B: Are community-associated methicillin-resistant Staphylococcus aureus (MRSA) strains replacing traditional nosocomial MRSA strains? Clin Infect Dis. 2008; 46(6): 787–94. PubMed Abstract \| Publisher Full Text\n\nHuang H, Flynn NM, King JH, et al.: Comparisons of community-associated methicillin-resistant Staphylococcus aureus (MRSA) and hospital-associated MSRA infections in Sacramento, California. J Clin Microbiol. 2006; 44(7): 2423–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAruhomukama D: DA_PRISMA 2009 checklist_2.doc. figshare. Online resource. 2020. http://www.doi.org/10.6084/m9.figshare.12563318.v1" }	[ { "id": "71664", "date": "20 Oct 2020", "name": "Fredrick Haraka", "expertise": [ "Reviewer Expertise Infectious disease", "evidence synthesis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript has been reviewed in consideration of the PRISMA guideline and checklist\nMboowa G et al., aimed at investigating anti-microbial resistance transmission dynamics in a hospital setting by pulling existing evidence through systematic review and meta-analysis. Mboowa G et al., searched two databases and restricted their search to Uganda. They found that Escherichia coli and Klebsiella pneumoniae were the most prevalent Enterobacteriaceae species and these species account for the highest proportions of extended-spectrum β-lactamase producers among gram-negative bacteria. Staphylococcus aureus was the most prevalent. The authors' topic is relevant for both patient and policy decisions, however, the authors will need to revise the manuscript before it can be accepted. Specifically;\n\nMajor comments:\nThe authors will need to redo and expand their search of relevant articles, indicate the time bound of the search and all relevant databases. The description of the search strategy must be detailed to include all components of the PICO question they intend to address.\n\nThe authors will need to adhere to the PRISMA guideline and rewrite the manuscript accordingly.\n\nI’m afraid the results do not reflect the analysis planned. I did not see any meta-analysis. The two tables presented do not show any results of pulled data.\n\nReviews are meant to provide a summary effect of an intervention/approach/test/drug etc. While it is important to define the PICO question clearly, restricting to a particular geographical region e.g. Uganda may unlikely provide the expected ‘global effect” of a review and meta-analysis. The authors might want to expand on this.\n\nMinor comments\nIn their title, the authors have indicated that they conducted a meta-analysis, however, it is disturbing to see that the authors did not report any summary measures in the abstract.\n\nAlthough the authors have included key summary structure, the information is definitely not exhaustive. The authors might want to look into the PRISMA checklist for guidance.\n\nThe authors might want to rewrite their introduction in a manner that allows the reader to explicitly understand why this review was necessary. The authors have spent a substantial amount of time discussing mechanisms of anti-microbial resistance and little on current evidence and critically highlight the gap this review is expected to fill. Specifically, a reader does not clearly get the PICO question being addressed.\n\nThe authors might want to mention whether or not there were any language restrictions.\n\nNo mention of if there was an existing protocol that was published before the actual review is conducted. It is important for the reader to know if any part of the initial planned objectives or analysis changed and why.\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? Partly", "responses": [] }, { "id": "95660", "date": "18 Oct 2021", "name": "Iryna Boiko", "expertise": [ "Reviewer Expertise Antimicrobial resistance", "antimicrobial surveillance" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDr. Gerald Mboowa et al. described the antimicrobial resistance transmission at referral hospitals in Uganda. The authors intended to use a meta-analysis of cross-sectional studies. The present manuscript highlighted a critical problem in Uganda and globally.\nHere I would like to provide several suggestions to improve the manuscript.\nMajors:\nMethods, Search strategy: Authors wrote: \"We also sought additional studies through screening the references of the selected studies.\" Would you please explain why authors needed to add additional studies by screening references of previously selected studies from Pubmed and Embase? Why have other studies not been found by Pubmed and Embase? This extended selection looks hard to track and, therefore, to reproduce.\n\nMethods: Authors wrote: \"Studies with the requirements above that had been published within the last ten years.\" Would you please add the period covered by the study for clarity? Has any language requirement been used for eligibility criteria?\n\nTables 1 and 2: Please indicate the data related to Uganda and add the study period. Please specify the study period for data in the columns \"Study 1-4\". As the authors provided data in rates, adding the confident intervals for the percentage data would be valuable.\n\nAs the authors provided meta-analysis, is it possible to summarize the antimicrobial resistance rates for the whole studied period?\n\nDiscussion: It would be valuable to compare bacterial etiology and antimicrobial susceptibility profiles from Uganda and globally for a similar period of studies.\n\nDiscussion: Please add the information about the study's limitations and their possible implication for the results.\nMinors\nDiscussion: Please add the references for the sentences:\n(4.1) \"According to the 2017 World Health Organization list of priority pathogens, these Enterobacteriaceae species have been identified to belong to the priority one or critical category, and particularly when these are producers of extended-spectrum β-lactamases\".\n\n(4.2) \"According to the 2017 World Health Organization list of priority pathogens, Staphylococcus aureus, has been identified to belong to the priority two or high category, and particularly when these are producers of extended-spectrum β-lactamases or which exhibit methicillin-resistance (MRSA)\".\n\nIntroduction: Please provide the acronym for the \"Extended-spectrum β-lactamase\" after it first appeared in the manuscript. Please note that this term is used 17 times in the manuscript, but the acronym was used once (Results section) through the text.\n\nMethods, Statistical analysis: Please provide the details for the IBM SPSS Statistics (version 16) according to the recommendation by Gouda, 20151, i.e. \"'SPSS Statistics for Windows, version x.0 (SPSS Inc., Chicago, Ill., USA)'\".\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": [] } ]	1	https://f1000research.com/articles/9-878
https://f1000research.com/articles/9-868/v1	31 Jul 20	{ "type": "Brief Report", "title": "Estimation of time-varying reproduction numbers of COVID-19 in American countries with regards to non-pharmacological interventions", "authors": [ "Fred G. Manrique-Abril", "Cristian Téllez-Piñerez", "Mario Pacheco-López", "Cristian Téllez-Piñerez", "Mario Pacheco-López" ], "abstract": "This study aimed to estimate the basic reproduction number and the time-varying estimate of effective reproductive number of COVID-19 in American countries as they implemented non-pharmacological strategies for the containment of the SARS-CoV-2 virus. Data sources included COVID-19 epidemic data from Johns Hopkins University’ data repository and official websites of countries with a relatively high incidence of COVID-19. The maximum likelihood method was used to estimate the and . The results showed that El Salvador, the Dominican Republic, Panama, and Peru have the lowest, while the USA and Canada have the highest. Other American countries have an around 1.4. Countries could be divided into three groups based on the varied behavior of over time. The first group (Mexico, USA, Colombia and Brazil) started with a high, which decreased post-intervention. In the second group, the intervention was performed at the moment when the, is high and it decreased slowly post-intervention (Canada, Argentina, Chile Peru, Panama and Dominican Republic). In the third group (Bolivia, Peru and Guatemala), the, was erratic and could not be attributable to the intervention. There is a close relationship between and non-pharmacological interventions decreed by governments of countries for the control of the COVID-19 pandemic. There are also immediate changes in the behavior of the indicator, and therefore the progression of the outbreak, when the interventions were implemented closer to the index case for each country.", "keywords": [ "Reproduction number", "effective reproductive number", "COVID-19", "American countries" ], "content": "Introduction\n\nOn January 21, the first case of COVID-19 in the United States1 was announced in a traveler who had arrived in the country from China 5 days earlier. Wuhan, China is where the health emergency originated2 in December 2019. From late January, the North American continent started to see case reports in different states, with cases arriving in South America on February 26 through Brazil after passing through Central America and the Caribbean3. Today, America is the epicentre of the pandemic, with the USA and Brazil being the two largest countries with the most cases. In addition, Peru, Chile, Mexico, Canada, Colombia and Ecuador are among the top 25 countries with the highest incidence worldwide.\n\nSince then, governments across the region have implemented a series of non-pharmacological measures to protect their citizens and contain the spread of COVID-19. All South American countries, including the French overseas region of French Guiana and the Falkland Islands, have reported the presence of coronavirus within their borders, and all took measures at different times of the outbreak.\n\nNon-pharmacological measures of containment (mitigation and suppression) have been implemented from the index case on different dates in each country2,4–6. This is based on projections of contagion and lethality resulting from SIR epidemiological models (susceptible, infected and recovered)7 with data taken from the behavior of the virus in Asia and Europe especially, and with approximations to the basic reproduction number (R0)8,9, effective reproduction number (Re), and variable time estimation of Re (Rt), among others. R0 estimates the speed with which a disease can spread in a population and plays a crucial role in predicting the prevalence of infectious disease, in addition to better understanding an epidemic outbreak and preparing the corresponding public health response. Rt is the reproduction number at time t, which reveals the actual transmission rate of the virus at a given moment. This number will vary depending on the control protocols established in each country.\n\nMitigation measures are aimed at slowing down the spread of infection by progressively reducing R010. Suppression measures aim to reduce R0 below 1, to progressively decrease the number of cases until they disappear. The greatest challenge that this strategy entails is that it needs to be maintained until the pandemic has disappeared or an effective treatment or vaccine is available6,10,11. The estimation of R0 and Rt in different scenarios allows a better prognosis of the trends of the global epidemic12, in addition to evaluating the effectiveness in reducing transmission with the measures taken by the different countries to contain COVID-1911.\n\nWhile this article was being prepared, the R0 and Rt values of some countries were published and modeled in three intervention scenarios; however, they only included the United States and Argentina from the American continent, limiting the comparative analysis for the characteristics of America13. Previous work has calculated the R0 and Rt using the maximum likelihood method and sequential Bayesian method, and found that European and North American countries possessed a higher R0 and unsteady Rt fluctuations, whereas some heavily affected Asian countries showed a relatively low R0 and declining Rt , with a small number of patients in Africa and Latin America, yet with the potential risk of large outbreaks. Xu et al.13 who determined Rt as an indicator to measure the transmission of SARS-CoV-2 before and after interventions, showed that the change in the rate of transmission of SARS-CoV-2 is associated with reducing social interactions.\n\nThis study aimed to estimate R0 and Rt in multiple countries of the American continent and evaluate their behavior from the first (index) case in each country, and the time of implementation of the chosen non-pharmacological containment measures.\n\n\nMethods\n\nCOVID-19 epidemic data were obtained from the Johns Hopkins University data repository (https://coronavirus.jhu.edu/). This information was verified using official country websites, and these websites were also used to collect data about non-pharmacological interventions (Table 1). We selected countries with a relatively high incidence of COVID-19, as follows: Argentina, Bolivia, Brazil, Canada, Colombia, Chile, the Dominican Republic, Ecuador, El Salvador, Guatemala, Honduras, Mexico, Panama, Peru, and the USA. Data were collected from the date on which the first case was reported in the country to June 12, 2020.\n\nWe used the COVID-19 daily incidence to estimate the basic reproduction number (R0) and the time-varying reproduction number (Rt) with packages R014 and EpiEstim15 on R software (version 4.0.1), respectively. Rt was calculated using a serial interval (SI) with a mean of 7.5 and a standard deviation of 3.42. The incubation period used was 5 days, taking into account the mean incubation period in 16 of 5.0 days (95% CI, 4.2–6.0), the median incubation period estimated in 17 of 5.1 days (95% CI, 4.5–5.8 days), and the mean incubation period in 2 of 5.2 days (95% CI, 4.1–7.0). We used the maximum likelihood method to estimate both R0 and Rt.\n\n\nResults\n\nEl Salvador, the Dominican Republic, Panama, and Peru have the lowest R0; in contrast, the USA and Canada have the highest. Other countries have an R0 around 1.4 (Figure 1).\n\nCountries could be divided into three groups based on the varied behavior of Rt over time (Figure 2–Figure 4). The first group (Mexico, USA, Colombia and Brazil) started with a high Rt, which decreased post-intervention. In the second group (Canada, Argentina, Chile Peru, Panama and Dominican Republic), the intervention was performed at the moment when the Rt, is high and this then decreased slowly post-intervention. In the third group (Bolivia, Peru and Guatemala), the Rt is erratic and cannot be attributable to the intervention; the Rt increases and decreases without any explanation.\n\nAnalysis by regions (North, Central and South America) shows differences between regions and countries. The countries of North America have a low Rt, which increased over time. Post-intervention control is achieved by a progressive decrease in all cases in Canada, Mexico and the USA. The behavior of the Dominican Republic and the countries of Central America is similar. Excepting Bolivia, Guatemala and Peru, the countries of South America and Central America show a similar behavior to the first group; the Rt is high at the beginning and decreases suddenly after control measures. However, Peru had a considerable peak after intervention implementation, as did Chile and Panama. This is possibly related to the flexible measures of mass community isolation\n\nDespite the differences between the first and second group, the Rt is high before the intervention in all countries and its decrease is achieved post-intervention, corroborating the purpose of these interventions in the mitigation and suppression of a pandemic.\n\nThe dotted horizontal line shows the target of 1 at Rt, and the vertical line represents the start date of the containment measures decreed in each country. Although there is a difference in the days, there is also a difference in the size of the countries, both in surface area and number of inhabitants that would allow mobility and different behavior of virus spread.\n\nThe dotted horizontal line shows the target of 1 at Rt, and the vertical line represents the start date of the containment measures decreed in each country. Although there is a difference in the days, there is also a difference in the size of the countries, both in surface area and number of inhabitants that would allow mobility and different behavior of virus spread.\n\nThe dotted horizontal line shows the target of 1 at Rt, and the vertical line represents the start date of the containment measures decreed in each country. Although there is a difference in the days, there is also a difference in the size of the countries, both in surface area and number of inhabitants that would allow mobility and different behavior of virus spread.\n\n\nDiscussion\n\nThe for the countries selected in this study was 1.2–1.8, which is much lower compared to China where it was reported to be 2.2, 2.6, 3.8, or even 6.47 according to different research2,18. Our study findings agree with Xu et al.13 in the estimated for the United States and Argentina. The Rt for these countries is also identical to that found by Xu et al.\n\nAs in the study by Xu et al.13, there are some limitations to the current study. First, the variation in depends on the series interval. Here, we assume 7.5 days for the 13 countries due to limited information on the disease in different nations13. In addition, we noticed some errors in the John Hopkins University data when we cross referenced these with official country websites. We were able to correct these by searching for cases directly on the official website of each country. Another drawback is the diversity of RT-PCR (real-time reverse transcription polymerase chain reaction) tests used in the diagnostic process and their sensitivity, which may throw up false negatives, perhaps under-estimating the true impact of the pandemic in various countries. Although data were available from other countries, in the case of Ecuador, the report was not included, because cases were not confirmed by RT-PCR, which is a that s technical criteria.\n\nThe epidemic trends in different regions around the world are significantly different due to the great difference in the design and implementation of prevention and control measures5. Estimates of both R0 and Rt in different countries will enable a better forecast of global epidemic trends.\n\nIn conclusions, our study shows that there is a close relationship between Rt and non-pharmacological interventions decreed by countries’ governments for the control of the COVID-19 pandemic. Additionally, we showed that there are immediate changes in the behavior of the Rt, and therefore in the progression of the outbreak, when the interventions are implemented closer to the index case for each country.\n\n\nData availability\n\nZenodo: DataStatistic/COVID19RtAme: Estimation of time-varying reproduction numbers of COVID-19 in American countries, http://doi.org/10.5281/zenodo.395796719.\n\nThis project contains the following underlying data:\n\nDataset and data dictionary\n\nR analysis code\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nTelemundo.com: El coronavirus llega a Estados Unidos reportan el primer caso de la enfermedad cerca de Seattle Telemundo. Telemundo.com. 2020; 1. Reference Source\n\nLi Q, Guan X, Wu P, et al.: Early transmission dynamics in Wuhan, China, of novel coronavirus-infected pneumonia. N Engl J Med. 2020; 382(13): 1199–1207. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHorwitz L, Nagovitch P, Sonneland HK, et al.: Where is the coronavirus in Latin America? AS/COA - Americas Society Council of the Americas. 2020. Reference Source\n\nMarkel H, Stern AM, Navarro JA, et al.: Non-pharmaceutical influenza mitigation strategies, US communities, 1918–1920 pandemic. Emerg Infect Dis. 2006; 12(12): 1961–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarcia LP, Duarte E: Intervenções não farmacológicas para o enfrentamento à epidemia da COVID-19 no Brasil. Epidemiol e Serv saude Rev do Sist Unico Saude do Bras. 2020; 29(2): e2020222. PubMed Abstract \| Publisher Full Text\n\nPrem K, Liu Y, Russell TW, et al.: The effect of control strategies to reduce social mixing on outcomes of the COVID-19 epidemic in Wuhan, China: a modelling study. Lancet Public Health. 2020; 5(5): e261–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nManrique-Abril FG, Agudelo-Calderon CA, González-Chordá VM, et al.: SIR model of the COVID-19 pandemic in Colombia. Rev Salud Pública. 2020; 22(2): 1–9. Reference Source\n\nGuerra FM, Bolotin S, Lim G, et al.: The basic reproduction number (R0) of measles: a systematic review. Lancet Infect Dis. 2017; 17(12): e420–8. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization (WHO): Report of the WHO-China Joint Mission on Coronavirus Disease 2019 (COVID-19). WHO-China Jt Mission Coronavirus Dis 2019. 2020; 1: 40. Reference Source\n\nGonzález-Jaramillo V, González-Jaramillo N, Gómez-Restrepo C, et al.: Impact of the COVID-19 pandemic on the Colombian population according to mitigation measures. Preliminary data from epidemiological models for the period March 18 to April 18, 2020. Rev Salud Pública. 2020; 22(2): 1–6. Reference Source\n\nFerguson NM, Laydon D, Nedjati-Gilani G, et al.: Impact of non-pharmaceutical interventions (NPIs) to reduce COVID-19 mortality and healthcare demand. ImperialAcUk. 2020; 3–20. Publisher Full Text\n\nRidenhour B, Kowalik JM, Shay DK: El número reproductivo básico (R0): Consideraciones para su aplicacioén en la salud pública. Am J Public Health. 2018; 108(Suppl 6): S455–65. Publisher Full Text \| Free Full Text\n\nXu C, Dong Y, Yu X, et al.: Estimation of reproduction numbers of COVID-19 in typical countries and epidemic trends under different prevention and control scenarios. Front Med. 2020; 1–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nObadia T, Haneef R, Boëlle PY: The R0 package: a toolbox to estimate reproduction numbers for epidemic outbreaks. BMC Med Inf Decis Mak. 2012; 12: 147. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCori A, Ferguson NM, Fraser C, et al.: A new framework and software to estimate time-varying reproduction numbers during epidemics. Am J Epidemiol. 2013; 178(9): 1505–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLinton NM, Kobayashi T, Yang Y, et al.: Incubation period and other epidemiological characteristics of 2019 novel coronavirus infections with right truncation: A statistical analysis of publicly available case data. J Clin Med. 2020; 9(2): 538. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLauer SA, Grantz KH, Bi Q, et al.: The incubation period of coronavirus disease 2019 (COVID-19) from publicly reported confirmed cases: Estimation and application. Ann Intern Med. 2020; 172(9): 577–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRead JM, Bridgen JR, Cummings DA, et al.: Novel coronavirus 2019-nCoV: early estimation of epidemiological parameters and epidemic predictions. medRxiv. 2020. Publisher Full Text\n\nMario PL, Cristian TP, Fred MA: DataStatistic/COVID19RtAme: Estimation of time-varying reproduction numbers of COVID-19 in American countries (Version v.1.0.1). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3957967" }	[ { "id": "68563", "date": "11 Aug 2020", "name": "Efrain Riveros-Perez", "expertise": [ "Reviewer Expertise Anesthesiology", "critical care", "business in healthcare" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nEnglish grammar is very poor. It impedes adequate understanding of the content. I suggest that the authors use a professional editing service.\n\nIn the title the term \"America\" is ambiguous. I suggest changing it by North America, Latin America and the Caribbean.\n\nThe manuscript is pertinent. However, the discussion needs more depth, contrasting to other authors' views. Please refrain from making value judgements.\n\nThe manuscript needs major changes before being considered for indexing.\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? 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": "6127", "date": "18 Nov 2020", "name": "Fred Manrique-Abril", "role": "Author Response", "response": "Thanks for your contributions, in a future article we will make improvements to achieve an outstanding product. The pandemic data is changing daily and other groups may model the updated Rt." }, { "c_id": "10254", "date": "20 Sep 2023", "name": "Fred Manrique-Abril", "role": "Author Response", "response": "Thanks for your review." } ] } ]	1	https://f1000research.com/articles/9-868
https://f1000research.com/articles/9-267/v1	17 Apr 20	{ "type": "Research Article", "title": "Bio-electrosprayed human neural stem cells are viable and maintain their differentiation potential", "authors": [ "Citlali Helenes González", "Suwan N. Jayasinghe", "Patrizia Ferretti", "Suwan N. Jayasinghe" ], "abstract": "Background: Bio-electrospray (BES) is a jet-based delivery system driven by an electric field that has the ability to form micro to nano-sized droplets. It holds great potential as a tissue engineering tool as it can be used to place cells into specific patterns. As the human central nervous system (CNS) cannot be studied in vivo at the cellular and molecular level, in vitro CNS models are needed. Human neural stem cells (hNSCs) are the CNS building block as they can generate both neurones and glial cells. Methods: Here we assessed for the first time how hNSCs respond to BES. To this purpose, different hNSC lines were sprayed at 10 kV and their ability to survive, grow and differentiate was assessed at different time points. Results: BES induced only a small and transient decrease in hNSC metabolic activity, from which cells recovered by day 6, and no significant increase in cell death was observed, as assessed by flow cytometry. Furthermore, bio-electrosprayed hNSCs differentiated as efficiently as controls into neurones, astrocytes and oligodendrocytes as shown by morphological, protein and gene expression analysis. Conclusions: This study highlights the robustness of hNSCs and identifies BES as a suitable technology that could be developed for the direct deposition of these cells in specific locations and configurations.", "keywords": [ "bio-electrospray", "death", "differentiation", "human", "in vitro", "neural stem cells", "neurone", "survival" ], "content": "Introduction\n\nElectrospraying is a very useful technique for fabricating micro- and nano-structures of different composition, texture and shape using a wide range of materials and cells. When cells are electrosprayed, the technique is known as bio-electrospray (BES). BES consists of a jet-based delivery system connected to an electric field that has the ability to generate nano-sized and cell-laden microdroplets (Jayasinghe & Townsend-Nicholson, 2006; Jayasinghe, 2011; Poncelet et al., 2012). This is due to the difference in electric potential between the charged needle and the ground electrode that forms an electric field, accelerating the charged cell suspension within the needle and forming an unstable jet. This methodology has the advantage of having the potential to achieve single-cell delivery, giving a more homogeneous cell distribution within a 3-dimensional (3D) construct, as well as being very suitable for microencapsulation (Boda et al., 2018; Jayasinghe, 2011; Poncelet et al., 2012). The configuration needed to obtain micro to nano-sized droplets and a near mono-distribution can be achieved by adjusting BES conditions (Jayasinghe et al., 2006; Jayasinghe & Townsend-Nicholson, 2006). Other strategies adopted to obtain such a high resolution require small diameter needles, resulting in shear stress to the cells and an inability to process high-density and/or viscous cell suspensions (Greig & Jayasinghe, 2008; Hall et al., 2008).\n\nAn important consideration for cell-based applications is that the high voltages and spraying action could have an adverse effect on the cells, and this may differ among cell types. Although it has been demonstrated that BES does not significantly affect a range of mammalian cells, and even small organisms, its effect have never been studied on human neural stem cells (hNSCs), the building block of the nervous system (Clarke & Jayasinghe, 2008; Geach et al., 2009; Hong et al., 2010; Jayasinghe et al., 2011; Joly et al., 2009; Tezera et al., 2017).\n\nhNSCs either derived from the embryonic nervous system or differentiated from pluripotent stem cells provide an ideal source for modelling the human nervous system. hNSCs have the capacity to self-renew and differentiate into the major cell types of the brain, neurones and glia (oligodendrocytes and astrocytes), and hold the potential to repair damaged tissue in the central nervous system (CNS) (Bianco & Robey, 2001; Gage & Temple, 2013). This makes them invaluable for the development of 3D models for the study of normal and abnormal developmental mechanisms, neurodegenerative disorders, neural repair and high-throughput screening of putative neuroactive drugs (Breier et al., 2010; Gage & Temple, 2013; Gu et al., 2016). There is also much interest in using hNSC to develop 3D systems for transplantation into the damaged CNS (Somaa et al., 2017; Vishwakarma et al., 2014).\n\nGiven the encouraging results from a few studies on mouse neural cells and human astrocytoma (Eagles et al., 2006; Eddaoudi et al., 2010; Jayasinghe & Townsend-Nicholson, 2006; Mongkoldhumrongkul et al., 2009a), we wished to establish whether hNSCs could be bio-electrosprayed, and specifically assess whether the procedure affected their survival and ability to undergo multi-lineage differentiation. Extensive analysis of hNSC survival/death and differentiation showed that hNSCs withstand the BES procedure vey well and could successfully differentiate towards neuronal, astrocyte and oligodendrocyte lineages with no alteration in gene expression following neuronal differentiation. Together, this study demonstrates that hNSCs remain viable over prolonged periods post-treatment and are capable of withstanding the pressure and stresses of being handled as high-density cell suspensions within a needle at a high voltage.\n\n\nMethods\n\nUnless otherwise indicated, chemicals were purchased from Sigma-Aldrich (UK). Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 GlutaMAXTM (DMEM/F12), Neurobasal®-A Medium, foetal bovine serum (FBS), N-2 (100x) and B-27 (50x) supplements were from Gibco, FGF-2, EGF and PDGF-aa from Peprotech, propidium iodide (PI) from Invitrogen and Allophycocyanin (APC) Annexin V from BD Pharmingen.\n\nHuman brain embryonic tissue was provided by the Human Developmental Biology Resource (HDBR, http://www.hdbr.org/). All procedures using human tissue were carried out in accordance with the Human Tissue Act 2006 with informed consent (REC reference: 18/LO/0822) for study participation under ethical approval (NRES Committee London – Fulham, London, UK). The hNSC lines used in this study had been derived from embryonic brain tissue at Carnegie Stage (Cs)17 and Cs23, and grown on laminin, as previously described (Taylor et al., 2019; U et al., 2014; Vagaska et al., 2016). In brief, cells were seeded at a density of ~11,000 cells/cm2 and grown at 37°C in a humidified incubator with 5% CO2 in medium containing: Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) supplemented with 1% (v/v) penicillin/streptomycin, 1% (v/v) 100x N2, 2% (v/v) B27, 20 ng/ml FGF-2, 20 ng/ml EGF, 50 µg/ml, BSA fraction V, 5 µg/ml heparin and 10 µg/ml laminin.\n\nDifferentiation was induced when hNSCs had reached confluency, approximately 3 days after plating.\n\nNeuronal differentiation. After 10 days in a medium consisting of DMEM containing Glutamax supplemented with 1% penicillin/streptomycin, 10 µM forskolin, 5 mM KCl, 2 mM valproic acid, 1 µM hydrocortisone and 5 µg/ml insulin for 10 days, cells were maintained in with Neurobasal®-A Medium supplemented with 1% L-glutamine, 1% penicillin/streptomycin and 2% B27 for 18 days (4 weeks total differentiation time). Protocol adapted from Guasti et al. (2012).\n\nOligodendrocyte differentiation. hNSCs were first incubated in DMEM/F12 containing 1% penicillin/streptomycin, 1% N2, 10 nM forskolin, 10 ng/ml FGF-2 and 10 ng/ml PDGF-aa for 14 days, and then in DMEM/F12 medium supplemented with 1% penicillin/streptomycin, 1% N2, 30 ng/ml tri-iodothyronine, 200 µM ascorbic acid and 10 ng/ml PDGF-aa for 7 days. PDGF-aa was then removed and cell incubated for a further 2 weeks to allow maturation (5 weeks total differentiation time).\n\nAstrocytic differentiation. This was induced by incubating hNSCs in DMEM/F12 supplemented with 10% (v/v) FBS and 1% penicillin/streptomycin for 2 weeks.\n\nThe BES system consisted of a high-voltage power supply (Glassman Europe Ltd., FP-30, Tadley, UK.) with a syringe pump (Harvard Apparatus) holding a needle of 1.5-mm outer diameter (0.8–0.9 mm inner diameter). The voltage was set at 10 kV and the flow rate at 250 ml/h. The procedure was carried out inside a class II biosafety cabinet to ensure sterility. hNSC suspensions with a density of ~1.3×106 cells per ml were divided into 1 experimental and 2 control groups, all run in triplicate. The experimental hNSCs were taken to the the bio-electrospray facility, which was located in a different building, and sprayed (BES group). One control group was transported to the BES facility but not sprayed (TC), and the other was left in the tissue culture laboratory (LC). All groups were replated at the same time.\n\nThe live/dead staining was performed 24 hours after BES. Hoechst 33258 and propidium iodide dissolved in phosphate buffered saline (PBS) were added to the culture medium at final concentrations of 2 μg/ml and 5 μg/ml, respectively. After a 2-hour incubation, cells were viewed and imaged using an IX71inverted microscope from Olympus equipped with a Lumen 200 metal arc lamp (Prior Scientific) and a monochrome ORCA-R2 digital camera (Hamamatsu Corp.) All images were processed with Fiji software (Java 8 version) (Schindelin et al., 2012).\n\nCells viability/metabolic activity was assessed 1, 3 and 6 days after BES by the MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay. In brief, cells were incubated for 2 hours in medium containing 10% MTT (stock solution 5 mg/ml in DMSO), after which the absorbance was measured at 595 nm with a spectrophotometer (Multiscan FC ThermoScientific).\n\nFlow cytometry analysis was performed immediately after and 3 days after BES. Roughly 1×106 hNSCs per sample were resuspended in 500 µl of 1:100 APC-Annexin V conjugate:Annexin V binding buffer (10 mM HEPES, 150 mM NaCl, 5 mM KCl, 5 mM MgCl2 and 1.8 mM CaCl2 adjusted with NaOH to pH 7.4). Samples were kept at room temperature (RT) in the dark for 20 minutes before adding propidium iodide (PI) to a final concentration of 5 µg/ml. Stained cells were kept on ice until loading on a BD FACSCalibur TM to carry out flow cytometry analysis. Data was analysed using Kaluza 1.3 software. As a positive control, hNSCs were treated with 10 µm thapsigargin for 24 hours to induce cell death prior to flow cytometry\n\nCells were fixed with 4% (w/v) paraformaldehyde (PFA) pH 7.4 for 15 minutes at RT, rinsed in PBS (phosphate buffer saline) and incubated in blocking solution (10% FBS, 3% BSA and 0.2% TritonX-100 in PBS) for 1 hour at RT. Incubation with primary and secondary antibodies at the indicated dilutions (Table 1) was overnight at 4°C, and for 1 hour at RT, respectively. The nuclear stain Hoechst 33258 (2 μg/ml) was added to the secondary antibody solution. Cells were mounted with Citifluor (Citifluor Ltd). An IX71inverted microscope (Olympus) with a monochrome ORCA-R2 digital camera (Hamamatsu Corp.) was used to acquire images. All images were processed with Fiji software (Schindelin et al., 2012).\n\nRNA was extracted from cell pellets using RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Complementary DNA (cDNA) was prepared from 500 ng of extracted RNA using MMLV reverse transcriptase (Promega) following the manufacturers protocol. Reverse transcription reactions were performed using a PTC-100 thermal cycler (MJ Research, Inc.). The sequences of the primers and conditions used are shown in Table 2. PCR reactions were performed in a Veriti Thermal Cycler (Applied Biosciences). To exclude contamination of the reagents, no-template controls (NTC) where water instead of cDNA was included were run in each experiment. A cDNA sample from a human embryonic brain (22 weeks post conception) was used as positive controls. Amplified products were separated by gel electrophoresis using 1.5% (w/v) agarose gels in tri-acetate EDTA (TAE) buffer and 1X SYBR Safe dye (ThermoFisher Scientific). Semi-quantification of the bands was performed using Fiji software (Schindelin et al., 2012) and the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), used to normalize expression.\n\nEach experiment was performed in biological triplicates unless stated otherwise. Statistical analysis was carried out with two-way ANOVA followed by Tukey’s multiple comparison test. Results are expressed as mean ± standard error of the mean. Differences were considered to be significant if p ≤ 0.05.\n\n\nResults\n\nWe first investigated whether hNSC viability is affected immediately after and at different times after bioelectrospraying (BES). Three groups were compared in all experiments: bio-electrosprayed hNSCs (BES) and two control groups, the BES control hNSCs (cells transported to the BES laboratory, but not sprayed; TC) and the tissue culture laboratory control hNSCs (cells not transported to the BES laboratory; LC).\n\nAt 24 hours after spraying, cells were double stained with Hoechst dye and PI to detect dead cells. No apparent difference in cell death between BES and control groups was observed by cell imaging (Figure 1A). To further assess the effect of BES on cell viability, hNSCs were labelled with PI and annexin V, a marker of apoptosis, immediately after spraying (Figure 1B) and 3 days later (Figure 1C). Cells were analysed by flow cytometry to detect early apoptotic (low PI and high APC-Annexin V), and late apoptotic/necrotic cells (high PI) as shown in Figure 1B, C. As summarized in Figure 1D, over 94% of cells were viable in all groups immediately after spraying (Day 0), and over 95% were viable at 3 days, with low levels of early apoptosis detected at both time points (1% and 0.5%, respectively). To establish whether BES affected cell behaviour over time, their metabolic activity, that reflects number of cells in the culture, was assessed by the MTT assay on day 1, 3 and 6 after BES. Metabolic activity increased over six days in all samples, although it was lower in the BES group than in the laboratory control at 1 and 3 days; however, by 6 days, no difference was observed among control and BES groups (Figure 1E). Together, these results suggest that BES does not negatively affect hNSCs viability over time.\n\nA) Staining with propidium iodide (PI, red) and Hoechst 33258 (blue) in live hNSCs (Cs 17, passage 22) 24 hours after spraying (BES) and in non-sprayed controls (TC: taken to the BES laboratory but non-sprayed; LC: not moved from the tissue culture laboratory). All pictures are at the same magnification. B–C) Analysis of Annexin V- and PI- positive cells by flow cytometry immediately after spraying (B) and 3 days after spraying (C). Representative scatter plots showing early apoptotic (EA), and apoptotic plus necrotic cell population (A&N) measured as percentages of total gated cells. D) Cell populations represented as percentages of total gated cells. No significant difference in the percentage of viable cells is observed between BES and controls (biological triplicates presented as mean ± SEM) as assessed by two way ANOVA. E) hNSC metabolic activity assessed by the MTT assay 1, 3 and 6 days after BES. Data represent mean absorbance ±SEM, n=6; * p ≤ 0.05, ** p ≤ 0.01 as assessed by two way ANOVA and Tukey’s multiple comparisons test.\n\nTo establish whether BES affected hNSCs differentiation capacity, two hNSCs lines were differentiated along the neuronal and glial lineages (astrocytes and oligodendrocytes). After 4 weeks of neuronal differentiation, processes that had started to grow at 10 days (see Extended data) had extended further in all groups, as shown by phase contrast images (Figure 2 and Extended data, Supplementary Figure S2) (Ferretti & Helenes González, 2020b). Immunofluorescence labelling for neuronal markers revealed comparable expression of the mature neuronal markers microtubule-associated protein 2 (MAP2), neurofilament 200 (NF200) and neuronal nuclear protein (NeuN) (Figure 2 and Extended data, Supplementary Figure S1 and S2) (Ferretti & Helenes González, 2020b) in the control and BES groups. In contrast, no significant expression of doublecortin (DCX), a marker of newly born and migrating neurons was detected in any group (Extended data, Supplementary Figure S1) (Ferretti & Helenes González, 2020b). These indicated that the BES process did not compromise hNSC neuronal differentiation.\n\nSprayed (BES) and control (TC: taken to the BES laboratory but non-sprayed; LC: not moved from the tissue culture laboratory) hNSCs (Cs 17, passage 22) differentiated for 4 weeks. Note typical neuronal morphology and neurite extension and expression of the neuronal markers, MAP2, microtubule-associated protein 2 (green), and NF200, neurofilament 200 (red). Nuclei are counterstained with Hoechst 33258 (blue). All phase contrast images are at the same magnification.\n\nUpon induction of astrocyte differentiation, hNSC morphology rapidly changed in both control and BES groups, with cells becoming more spread and flatter than in undifferentiated controls, consistent with astrocytic differentiation (Figure 3 and Extended data, Supplementary Figure S3) (Ferretti & Helenes Gonzalez, 2020b). This was further supported by expression of vimentin and glial fibrillary acidic protein (GFAP). Together, comparable morphological appearance and glial markers expression in all groups indicates that BES does not interfere with astrocytic differentiation of hNSCs.\n\nSprayed (BES) and control (TC: taken to the BES laboratory but non-sprayed; LC: not moved from the tissue culture laboratory) hNSCs (Cs 17, passage 22) differentiated for 2 weeks. Note the flatten morphology typical of astrocytes morphology and expression of astrocyte markers, Vimentin (green) and GFAP (glial fibrillary acidic protein; red). Nuclei are counterstained with Hoechst 33258 (blue). All phase contrast images are at the same magnification.\n\nFinally, the effect of BES on oligodendrocyte differentiation was tested. At 5 weeks of differentiation after BES, both control and BES cultures had acquired a branched morphology with long processes (Figure 4 and Extended data, Supplementary Figure S4) (Ferretti & Helenes González, 2020b). Labelling of oligodendrocyte progenitor markers revealed a few cells positive for the oligodendrocyte marker, O4 (Figure 4 and Extended data, Supplementary Figure S4) (Ferretti & Helenes González, 2020b), and several positive for A2B5, that is expressed in oligodendrocyte progenitor cells (Figure 4). There was no visible difference in the expression of these markers between BES cells and their controls, indicating that the BES process does not alter the oligodendrocyte differentiation.\n\nSprayed (BES) and control (TC: taken to the BES laboratory but non-sprayed; LC: not moved from the tissue culture laboratory) hNSCs (Cs 17, passage 22) differentiated for 5 weeks. Note the presence of cells with different morphologies with a few expressing the oligodendrocyte marker, O4, and a larger proportion the glial precursor marker, A2B5. Nuclei are counterstained with Hoechst 33258 (blue). All phase contrast images are the same magnification.\n\nThe effect of BES on neuronal differentiation was further investigated at the gene expression level in hNSCs after 4 weeks of neuronal differentiation. As mixed cultures are normally obtained following neuronal induction, rather than pure neuronal populations, both neuronal and glial markers were assessed to establish whether BES changed the balance of differentiation among these cell types. Transcripts for the neuronal markers, NF200, neuron-specific enolase (NSE) and MAP2, as well as the glial markers glutamate aspartate transporter (GLAST), Olig2 and GFAP were detected in all groups by RT-PCR (Figure 5A–C and Extended data, Supplementary Figure S5A–C) (Ferretti & Helenes González, 2020b). To obtain more quantitative information, gene expression was further investigated by semi-quantitative RT-PCR analysis in two hNSC lines (Figure 5D and Extended data, Supplementary Figure S5D) (Ferretti & Helenes González, 2020b). Relative expression of genes was normalised to GAPDH. No significant differences in glial marker expression between the control and BES groups was observed in either line. Also, neuronal markers were expressed at comparable levels, with the exception of NF200, which was expressed at slightly higher levels in the BES group in one of the cell lines (Extended data, Supplementary Figure S5D) (Ferretti & Helenes González, 2020b). Overall, the expression of all but one marker were unchanged, suggesting no significant effects resulted from BES.\n\nA–C. Expression of neuronal markers (A), glial markers (B), and a reference house-keeping gene, GAPDH, (C) in biological triplicates of sprayed (BES) and control (TC: taken to the BES laboratory but non-sprayed; LC: not moved from the tissue culture laboratory) hNSCs (Cs 17, passage 25). MAP2: microtubule-associated protein 2; NF200: neurofilament 200; NSE: neuron-specific enolase; GFAP: glial fibrillary acidic protein; GLAST: glutamate aspartate transporter; OLIG2: oligodendrocyte transcription factor 2; GAPDH: glyceraldehyde 3-phosphate dehydrogenase. +CTRL: human embryonic brain cDNA used as positive control; NTC: no template control using water instead of cDNA. D) Relative expression of neuronal and glial markers assessed by densitometry. Data are means ± SEM of band intensity normalised to GAPDH. No significant difference in gene expression is observed (two way ANOVA).\n\n\nDiscussion\n\nOnly a few studies on the effect of BES on human cells have been carried out; these focussed on human mesenchymal stem cells (MSCs), either primary or hTERT immortalized, and tumour cells (Braghirolli et al., 2013; Eddaoudi et al., 2010; Mongkoldhumrongkul et al., 2009a; Ye et al., 2015). In this study, to our knowledge we show for the first time that hNSCs can withstand the BES procedure without any negative effect on their self-renewal capacity and importantly on their neuronal and glial differentiation potential. The high voltage of 10kV used here, the pressure applied by the syringe pump, the flow rate, the high-density solution in a small-bore needle (0.8–0.9 mm) and the handling of the cells in a separate laboratory had limited impact on hNSCs. It is well established that high voltages can be detrimental to cells, for example when cells are electroporated (Traitcheva & Berg, 2010). However, the fact that BES operates at high voltage but low current, in the nano-ampere range (Pakes et al., 2011; Poncelet et al., 2012), could help explain why cells do not show adverse effects when subjected to high voltages. Recently, a study assessing the effect of bioprinting on Schwann cells and myoblasts has suggested that this technique affects their viability and proliferative activity (Ning et al., 2018). Therefore, at least for some cell types, BES could provide a valuable alternative to bioprinting.\n\nOur findings on the safety of BES on hNSCs are consistent with findings in a number of cell types and organisms previously investigated, including mesenchymal cells (Hong et al., 2010; Irvine et al., 2007; Jayasinghe et al., 2011; Ye et al., 2015), immortalised mouse neural cells and human astrocytoma cells (Eagles et al., 2006; Eddaoudi et al., 2010; Jayasinghe & Townsend-Nicholson, 2006), and nematodes (Mongkoldhumrongkul et al., 2010). In all these studies, a survival of up to 90% after BES was observed. Notably, the hNSCs displayed an even higher survival rate (>94%), suggesting that these human stem cells are more robust than most of the cells previously studied.\n\nAnother difference between previous studies and ours is the length of time we monitored the cells for. Here, not only we measured metabolic activity up to 6 days, but also monitored the cells over weeks in the differentiation experiments, where undifferentiated controls were run in parallel. The use of two controls also showed that an initial small decrease in metabolic activity on BES samples was partly due to the transfer of the samples to the BES laboratory, and that full recovery had occurred by day 6. Viability of rabbit bone marrow-derived MSCs after BES was lower than that of hNSCs, with a metabolic/proliferation rate significantly lower than in controls even at a lower bio-electrospraying voltage than that used in our study (Sahoo et al., 2010). Together, the combination of cell death and survival assays and long-term monitoring used here provides clear evidence that BES does not affect hNSCs viability either immediately or over time.\n\nOur tri-lineage differentiation study has clearly shown no changes in hNSC differentiation potential after BES. As from previous reports, hNSC induction of neuronal differentiation resulted in a heterogeneous population of cells, and this was comparable across groups as shown by mRNA expression (Glaser et al., 2007; Gu et al., 2016; Sun et al., 2008). Astrocytic differentiation resulted in a more homogeneous population and all cells expressed vimentin and GFAP, though at different extents. The low number of cells positive for the oligodendrocyte marker O4 upon induction of oligodendrocyte differentiation is comparable in control and BES groups, and consistent with the long time required for human oligodendrocyte maturation. Indeed, a marker of less mature cells, A2B5, was expressed in a much higher proportion of cells.\n\nThis again supports the view that hNSCs are very resistant to external stimuli. This may be a property of human stem cells or of neural stem cells, or both, and extensive investigation will be required to compare stem cell types across species to clarify this issue. Rabbit bone marrow-derived MSCs have been shown to maintain differentiation potential along three mesenchymal lineages after BES at a lower voltage than the one used here (Sahoo et al., 2010). By contrast, human MSCs derived from human deciduous tooth pulp appear to better withstand BES even at higher voltage (15 kV), as well as maintain tri-lineage differentiation potential (Braghirolli et al., 2013; Braghirolli et al., 2015). Also human adipose-derived MSCs (ADSCs) survived and differentiated efficiently after BES (Ye et al., 2015).\n\n\nConclusion\n\nAnalysis of cell viability, tri-lineage differentiation capacity and gene expression demonstrated that the BES process does not adversely affect hNSCs either in the short or long term. Notably, it highlighted the robustness of these human stem cells. In conclusion, this study shows that BES is a suitable tool for the direct handling of hNSCs. Therefore, it may provide a suitable technology for deposition of hNSCs to specific locations in damaged nervous system in vivo or within suitable scaffolds for neural tissue engineering. Furthermore, this approach could be developed to generate well-controlled human neural 3D models for studying neural development or disease and responses to putative novel therapeutic interventions.\n\n\nData availability\n\nHarvard Dataverse: Bio-electrosprayed human neural stem cells are viable and maintain their differentiation potential- Underlying data of main figures. https://doi.org/10.7910/DVN/CAASEG (Ferretti & Helenes González, 2020a).\n\nThis project contains the raw uncropped images used to produce each figure, in addition to flow cytometry, cell viability and RT-PCR output data.\n\nHarvard Dataverse: Bio-electrosprayed human neural stem cells are viable and maintain their differentiation potential- Underlying data of supplementary figures. https://doi.org/10.7910/DVN/CLGEWR (Ferretti, 2020).\n\nThis project contains the raw uncropped images used to produce each of the supplementary figures (see Extended data), in addition to RT-PCR output data for Supplementary Figure S5D.\n\nHarvard Dataverse: Bio-electrosprayed human neural stem cells are viable and maintain their differentiation potential- Extended data. https://doi.org/10.7910/DVN/M8ZFNR (Ferretti & Helenes González, 2020b).\n\nThis project contains the file ‘Supplementary figures.pdf’, which contains the following extended data:\n\nFigure S1 Expression of neuronal markers in hNSCs after 4 weeks of differentiation. (A–B) Neuronal nuclear protein (NeuN) and doublecortin (DCX) in sprayed (BES) and control (TC: taken to the BES laboratory but non-sprayed; LC: not moved from the tissue culture laboratory) in two hNSC lines, Cs 17, passage 22 (A) and Cs23, passage 20 (B). Nuclei are counterstained with Hoechst 33258 (blue).\n\nFigure S2. Neuronal differentiation of hNSCs after bio-electrospray assessed by phase contrast imaging and double-labelling for neuronal markers. Sprayed (BES) and control (TC: taken to the BES laboratory but non-sprayed; LC: not moved from the tissue culture laboratory) hNSCs (Cs 23, passage 20) differentiated for 4 weeks. Note typical neuronal morphology and neurite extension and expression of the neuronal markers, MAP2, microtubule-associated protein 2 (green), and NF200, neurofilament 200 (red). Nuclei are counterstained with Hoechst 33258 (blue). All phase contrast images are at the same magnification.\n\nFigure S3. Astrocyte differentiation of hNSCs after bio-electrospray assessed by phase contrast imaging and double-labelling for astrocyte markers. Sprayed (BES) and control (TC: taken to the BES laboratory but non-sprayed; LC: not moved from the tissue culture laboratory) hNSCs (Cs 23, passage 20) differentiated for 2 weeks. Note the flatten morphology typical of astrocytes morphology and expression of astrocyte markers, Vimentin (green) and GFAP (glial fibrillary acidic protein; red). Nuclei are counterstained with Hoechst 33258 (blue). All phase contrast images are at the same magnification.\n\nFigure S4. Oligodendrocyte differentiation of hNSC assessed after bio-electrospray by phase contrast imaging and immunostaining. Sprayed (BES) and control (TC: taken to the BES laboratory but non-sprayed; LC: not moved from the tissue culture laboratory) hNSCs (Cs 23, passage 20) differentiated for 5 weeks. Note the presence of cells with different morphologies with a few expressing the oligodendrocyte marker, O4, and a larger proportion the glial precursor marker, A2B5. Nuclei are counterstained with Hoechst 33258 (blue). All phase contrast images are the same magnification.\n\nFigure S5. Expression of neural markers in hNSCs neuronally differentiated for 4 weeks after bio-electrospray assessed by RT-PCR. (A-C) Expression of neuronal markers (A), glial markers (B), and a reference house-keeping gene (C) in biological triplicates of sprayed (BES) and control (TC: taken to the BES laboratory but non-sprayed; LC: not moved from the tissue culture laboratory) hNSCs (Cs 23, passage 22). MAP2: microtubule-associated protein 2; NF200: neurofilament 200; NSE: neuron-specific enolase; GFAP: glial fibrillary acidic protein; GLAST: glutamate aspartate transporter; OLIG2: oligodendrocyte transcription factor 2; GAPDH: glyceraldehyde 3-phosphate dehydrogenase. +CTRL: human embryonic brain cDNA used as positive control (22 weeks post conception); NTC: no template control using water instead of cDNA. (D) Relative expression of neuronal and glial markers assessed by densitometry. Data are means ± SEM of band intensity normalised to GAPDH. Increased NF200 expression (* p 0.05) is observed in the BES group (two way ANOVA with Tukey’s multiple comparisons test).\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Author contributions\n\n\n\nCHG designed and performed experiments, analysed data, obtained funding and wrote the manuscript; SNJ advised and provided training on bio-electrospray and reviewed the manuscript; PF planned research, obtained funding, and wrote the manuscript.\n\n\nAcknowledgements\n\nWe wish to thank Dr Dale Moulding at the ICH Microscopy Facility for his advice on image acquisition and Dr Ayad Eddaoudi for help with flow cytometry data acquisition.\n\n\nReferences\n\nBianco P, Robey PG: Stem cells in tissue engineering. Nature. 2001; 414(6859): 118–21. 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Harvard Dataverse, V2. 2020. http://www.doi.org/10.7910/DVN/CLGEWR\n\nFerretti P, Helenes González C: \"Bio-electrosprayed human neural stem cells are viable and maintain their differentiation potential- Underlying data of main figures\". Harvard Dataverse, V3. 2020a. http://www.doi.org/10.7910/DVN/CAASEG\n\nFerretti P, Helenes González C: \"Bio-electrosprayed human neural stem cells are viable and maintain their differentiation potential- Extended data\". Harvard Dataverse, V1. 2020b. http://www.doi.org/10.7910/DVN/M8ZFNR\n\nGage FH, Temple S: Neural stem cells: generating and regenerating the brain. Neuron. 2013; 80(3): 588–601. PubMed Abstract \| Publisher Full Text\n\nGeach TJ, Mongkoldhumrongkul N, Zimmerman LB, et al.: Bio-electrospraying living Xenopus tropicalis embryos: investigating the structural, functional and biological integrity of a model organism. Analyst. 2009; 134(4): 743–747. 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PubMed Abstract \| Publisher Full Text\n\nHall RP, Ogilvie CM, Aarons E, et al.: Genetic, genomic and physiological state studies on single-needle bio-electrosprayed human cells. Analyst. 2008; 133(10): 1347–1351. PubMed Abstract \| Publisher Full Text\n\nHong J, deMello AJ, Jayasinghe SN: Bio-electrospraying and droplet-based microfluidics: control of cell numbers within living residues. Biomed Mater. 2010; 5(2): 21001. PubMed Abstract \| Publisher Full Text\n\nIrvine S, Arumuganathar S, McEwan JR, et al.: Coaxial Aerodynamically Assisted Bio-jets: A Versatile Paradigm for Directly Engineering Living Primary Organisms. Eng Life Sci. 2007; 7(6): 599–610. Publisher Full Text\n\nJayasinghe SN: Bio-electrosprays: from bio-analytics to a generic tool for the health sciences. Analyst. 2011; 136(5): 878–890. PubMed Abstract \| Publisher Full Text\n\nJayasinghe SN, Eagles PA, Qureshi AN: Electric field driven jetting: an emerging approach for processing living cells. 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PubMed Abstract \| Publisher Full Text\n\nMongkoldhumrongkul N, Swain SC, Jayasinghe SN, et al.: Bio-electrospraying the nematode Caenorhabditis elegans: studying whole-genome transcriptional responses and key life cycle parameters. J R Soc Interface. 2010; 7(45): 595–601. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNing L, Betancourt N, Schreyer D, et al.: Characterization of Cell Damage and Proliferative Ability during and after Bioprinting. ACS Biomater Sci Eng. 2018; 4(11): 3906–3918. Publisher Full Text\n\nPakes NK, Jayasinghe SN, Williams RS: Bio-electrospraying and aerodynamically assisted bio-jetting the model eukaryotic Dictyostelium discoideum: assessing stress and developmental competency post treatment. J R Soc Interface. 2011; 8(61): 1185–1191. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPoncelet D, de Vos P, Suter N, et al.: Bio-electrospraying and cell electrospinning: progress and opportunities for basic biology and clinical sciences. Adv Healthc Mater. 2012; 1(1): 27–34. PubMed Abstract \| Publisher Full Text\n\nSahoo S, Lee WC, Goh JC, et al.: Bio-electrospraying: A potentially safe technique for delivering progenitor cells. Biotechnol Bioeng. 2010; 106(4): 690–698. PubMed Abstract \| Publisher Full Text\n\nSchindelin J, Arganda-Carreras I, Frise E, et al.: Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012; 9(7): 676–682. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSomaa FA, Wang TY, Niclis JC, et al.: Peptide-Based Scaffolds Support Human Cortical Progenitor Graft Integration to Reduce Atrophy and Promote Functional Repair in a Model of Stroke. Cell Rep. 2017; 20(8): 1964–1977. PubMed Abstract \| Publisher Full Text\n\nSun Y, Pollard S, Conti L, et al.: Long-term tripotent differentiation capacity of human neural stem (NS) cells in adherent culture. Mol Cell Neurosci. 2008; 38(2): 245–258. PubMed Abstract \| Publisher Full Text\n\nTaylor AC, Helenes González C, Ferretti P, et al.: Spontaneous differentiation of human neural stem cells on nanodiamond. Adv Biosyst. 2019; 3(4): 1800299. Publisher Full Text\n\nTezera LB, Bielecka MK, Chancellor A, et al.: Dissection of the host-pathogen interaction in human tuberculosis using a bioengineered 3-dimensional model. eLife. 2017; 6: pii: e21283. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTraitcheva N, Berg H: Electroporation and alternating current cause membrane permeation of photodynamic cytotoxins yielding necrosis and apoptosis of cancer cells. Bioelectrochemistry. 2010; 79(2): 257–260. PubMed Abstract \| Publisher Full Text\n\nU KP, Subramanian V, Nicholas AP, et al.: Modulation of calcium-induced cell death in human neural stem cells by the novel peptidylarginine deiminase-AIF pathway. BBA Mol Cell Res. 2014; 1843(6): 1162–1171. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVagaska B, New SE, Alvarez-González C, et al.: MHC-class-II are expressed in a subpopulation of human neural stem cells in vitro in an IFNγ-independent fashion and during development. Sci Rep. 2016; 6: 24251. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVishwakarma SK, Bardia A, Tiwari SK, et al.: Current concept in neural regeneration research: NSCs isolation, characterization and transplantation in various neurodegenerative diseases and stroke: A review. J Adv Res. 2014; 5(3): 277–294. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYe C, He Z, Lin Y, et al.: Bio-electrospraying is a safe technology for delivering human adipose-derived stem cells. Biotechnol Lett. 2015; 37(2): 449–456. PubMed Abstract \| Publisher Full Text" }	[ { "id": "62497", "date": "01 May 2020", "name": "Jamie A. Davies", "expertise": [ "Reviewer Expertise Tissue engineering & Synthetic Biology (but my reservations", "which are purely about more methodological details being needed in places", "do not depend on any deep 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\nOverall, this study is a report of useful, incremental research. Previous reports, including some by the same lead author, have shown that a range of mammalian cells tolerate BES well. This report effectively says “and so do hNSCs”. Not a surprise (there was no obvious reason that the wouldn't) but a useful thing to know nevertheless.\nSpecific comments:\nI liked your use of both transported and un-transported controls.\nIn the methods, it would be helpful to have catalogue numbers of all of the biologics (growth factors etc), because these can be purchased in subtly different forms (native, recombinant, etc). Knowing the exact one helps the cause of reproducibility.\nIn the first paragraph of the Introduction, 3 lines up, it would be helpful to know what BES conditions can be altered (otherwise it is hard to make the comparison with the less advantageous techniques mentioned in the subsequent sentence).\nMethods, 2nd paragraph, last line – what is N2? If it is the culture supplement that ThermoFisher (and maybe others) sell, please give a reference or at least an explanation.\nCan you say more about the needle? (eg supplier code) and the field strength (you keep quoting 10kV, but not the distance over which this potential exists). Again, this is for reproducibility.\nIt is also critical that you provide more information about the BES setup (distances etc); assuming this is one you have described in previous publications, you can just cite these publications. But without this information, nobody could reproduce this work.\nStrictly, the whole manuscript requires careful copy-editing to remove minor errors of punctuation and syntax, but these errors do not make the existing manuscript too hard to understand.\n\nIs the work 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": "5615", "date": "31 Jul 2020", "name": "Patrizia Ferretti", "role": "Author Response F1000Research Advisory Board Member", "response": "We are grateful to the Reviewer for his helpful comments. “In the methods, it would be helpful to have catalogue numbers of all of the biologics (growth factors etc), because these can be purchased in subtly different forms (native, recombinant, etc). Knowing the exact one helps the cause of reproducibility.”This has been included, but please note that full information about a couple of reagents is still missing because we do not have access to it due to the current lockdown to control the Covid-19 pandemic. “In the first paragraph of the Introduction, 3 lines up, it would be helpful to know what BES conditions can be altered (otherwise it is hard to make the comparison with the less advantageous techniques mentioned in the subsequent sentence).” The configuration needed to obtain micro to nano-sized droplets, and a near mono-distribution can be achieved by adjusting BES conditions to obtain a stable cone jet mode. The primary conditions to consider are viscosity and electrical conductivity of the sprayed liquid. Ideally, the cell-laden suspension should have high viscosity and low electrical conductivity. A coaxial arrangement with an outer needle carrying an encapsulating biomaterial and an inner needle carrying the cells in suspension could also be considered (Jayasinghe and Townsend-Nicholson, 2006). This has now been specified. “Can you say more about the needle? (eg supplier code) and the field strength (you keep quoting 10kV, but not the distance over which this potential exists). Again, this is for reproducibility. It is also critical that you provide more information about the BES setup (distances etc); assuming this is one you have described in previous publications, you can just cite these publications. But without this information, nobody could reproduce this work.”The needle used in these studies are similar to those used in our previous studies[x], and is a standard straight cut hypodermic needle made of stainless steel. The field strength was 0.2kv/mm (10kv over 50mm) and a reference has been added: O'Neill HC, Maalouf WE, Harper JC, Jayasinghe SN. Bio-electrosprayed human sperm remain viable. Materials Today. 2019; 1;31:21-30.“" } ] }, { "id": "62498", "date": "11 May 2020", "name": "Jingwei Xie", "expertise": [ "Reviewer Expertise Electrospray", "Biomaterials", "Tissue Regeneration" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 reports bioelectrospray of human neural stem cells and examines the survival, growth, and differentiation after bioelectrospray. This is an interesting work as bioelectrospray could be used to deposit human neural stem cells to form patterns for forming and studying complex neural tissue constructs. Overall, this work is satisfactory. However, authors should provide details of the bioelectrospray process. For example, how long for the process? In addition, the flow rate 250 ml/h seems too high. Please double check.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5616", "date": "31 Jul 2020", "name": "Patrizia Ferretti", "role": "Author Response F1000Research Advisory Board Member", "response": "We thank the Reviewer for this comment. It should be noted that this study focused on the initial crucial question concerning cell viability and maintenance of differentiation capability after bioelectrospraying rather than on the formation of the stable cone jet mode needed to obtain micro to nano-sized droplets. Hence, a high flow rate was used to evaluate cell viability when exposed to the high voltage and needle constraints of BES. Because of the high flow rate, cells were subjected to the voltage for less than a minute. This study provides the foundation for the use of hNSCs to refine the conditions for achieving a stable cone jet mode. To this purpose, flow rate adjustments, single or coaxial needle arrangement, and liquid properties mentioned will need to be analysed." } ] }, { "id": "62499", "date": "22 May 2020", "name": "Patricia Pranke", "expertise": [ "Reviewer Expertise Tissue engineering and regenerative medicine - area of stem cell and nanotechnology (electrospinning mainly)" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 analyzes the effect of the bio-electrospray (BES) technique on human neural stem cells (hNSCs). Although the BES provoked a decrease in hNSC metabolic activity, there was no increase in hNSC death. The differentiated assays were also successful, showing that the BES did not adversely affect the differentiation capacity of the hNSCs or their gene expression. The figures are very illustrative and very well executed.\n\nPlease correct VERY instead of “VEY well and could….” In the line 1 on the second column of the Introduction.\n\nThe paper is very well written and it could probably be indexed without additional revision. Having said that, there are a few minor errors which could be adjusted, but they are small details. I have 6 more additional examples if you need them.\n\nIn the Abstract:\n\n“Bio-electrospray (BES) is a jet based” – change to “Bio-electrospraying (BSE)”\n\n“…has the ability to form...” – “..has the ability of forming...”\n\n“...different hNSC lines..' – '...a number of hNSC lines...”\n\n“...from which cells recovered...” – “...from which the cells recovered...”\n\n“...as efficiently as controls…” – “...as efficiently as the controls…”\n\n“...and oligodentocytes as shown by...” – “...and oligodentrocytes, as shown by...”\n\nIs the work 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": "5617", "date": "31 Jul 2020", "name": "Patrizia Ferretti", "role": "Author Response F1000Research Advisory Board Member", "response": "We thank you the Reviewer for their kind comments and useful suggestion which we have incorporated in the revised version of the manuscript." } ] } ]	1	https://f1000research.com/articles/9-267
https://f1000research.com/articles/9-859/v1	31 Jul 20	{ "type": "Brief Report", "title": "Conscious prone positioning during non-invasive ventilation in COVID-19 patients: experience from a single centre", "authors": [ "Helmi C. Burton-Papp", "Alexander I. R. Jackson", "Ryan Beecham", "Matteo Ferrari", "Myra Nasim-Mohi", "Michael P. W. Grocott", "Robert Chambers", "Ahilanandan Dushianthan", "University Hospital Southampton Critical Care Team", "REACT COVID Investigators", "Helmi C. Burton-Papp", "Alexander I. R. Jackson", "Ryan Beecham", "Matteo Ferrari", "Myra Nasim-Mohi", "Michael P. W. Grocott", "Robert Chambers" ], "abstract": "Critically ill patients admitted to hospital following SARS-CoV-2 infection often experience hypoxic respiratory failure and a proportion require invasive mechanical ventilation to maintain adequate oxygenation. The combination of prone positioning and non-invasive ventilation in conscious patients may have a role in improving oxygenation. The purpose of this study was to assess the effect of prone positioning in spontaneously ventilating patients receiving non-invasive ventilation admitted to the intensive care. Clinical data of 81 patients admitted with COVID 19 pneumonia and acute hypoxic respiratory failure were retrieved from electronic medical records and examined. Patients who had received prone positioning in combination with non-invasive ventilation were identified. A total of 20 patients received prone positioning in conjunction with non-invasive ventilation. This resulted in improved oxygenation as measured by a change in PaO2/FiO2 (P/F) ratio of 28.7 mmHg while prone, without significant change in heart rate or respiratory rate. Patients on average underwent 5 cycles with a median duration of 3 hours. There were no reported deaths, 7 of the 20 patients (35%) failed non-invasive ventilation and subsequently required intubation and mechanical ventilation. In our cohort of 20 COVID-19 patients with moderate acute hypoxic respiratory failure, prone positioning with non-invasive ventilation resulted in improved oxygenation. Prone positioning with non-invasive ventilation may be considered as an early therapeutic intervention in COVID-19 patients with moderate acute hypoxic respiratory failure.", "keywords": [ "COVID-19", "Intensive Care", "Non-invasive ventilation", "Prone" ], "content": "Introduction\n\nThe critical illness characterised by the SARS-CoV-2 viral infection (Coronavirus disease 2019; COVID-19) often results in respiratory symptoms leading to acute hypoxic respiratory failure (AHRF) necessitating mechanical ventilation. Supplemental oxygen is the initial mainstay of therapy. Non-invasive ventilation (NIV) has been shown to be a successful alternative to mechanical ventilation in the early stages of the related coronavirus severe acute respiratory syndrome (SARS) and in COVID-19 without a subsequent need for invasive mechanical ventilation1,2. Despite COVID-19 being a novel single disease entity, various phenotypic variations based on biological markers of immunity, prothrombotic features, ventilator mechanics and radiological changes have been documented3. Consequently, the response to specific therapies may vary between COVID-19 patients with AHRF.\n\nProne positioning has been often adopted to improve gas exchange in mechanically ventilated patients with moderate to severe refractory hypoxia associated with acute respiratory distress syndrome (ARDS). Prone position was not associated with an improved outcome in a Cochrane review of ARDS patients receiving invasive mechanical ventilation4. However, subgroup analysis demonstrated a better outcome among those patient groups with severe ARDS. The Proning Severe ARDS (PROSEVA) multicentre randomised-controlled trial has demonstrated improved survival in patients with severe ARDS who had received early, long sessions of prone positioning when compared to the control group of supine patients5. Despite most COVID-19 patients with AHRF fulfilling the ARDS clinical definition, there are demonstrable variations in response to therapy between COVID-19 lung disease and ARDS. Although the use of prone position in COVID-19 AHRF has not been formally evaluated by clinical trials, its use is supported by several international guidelines, including The Surviving Sepsis Campaign and ANZICS COVID-19 guidelines6,7. Moreover, recently a study demonstrated improved oxygenation during a single episode of prone positioning in awake non-ventilated COVID-19 patients8, while another has demonstrated its feasibility in a non-critical care environment9. A recent review of conscious proning in ARDS and COVID-19 infection which included a handful of studies with small number of patients and concluded short term improvements in the oxygenation10. Here we build on this literature offering an examination of changes in oxygenation, as measured by PaO2/FiO2, across multiple episodes of prone positioning in conscious patients, with moderate to severe hypoxia, undergoing non-invasive ventilation following admission to the intensive care unit for advanced respiratory support.\n\n\nMethods\n\nWe collected data from all COVID-19 reverse transcriptase polymerase chain reaction (RT-PCR)-confirmed (nasal and throat swab specimens) admissions to the General Intensive Care Unit, University Hospital Southampton NHS Foundation Trust between 4th March 2020 and 11th May 2020. When clinical stability allowed, all patients presenting with AHRF needing additional respiratory support, beyond standard oxygen therapy, were trialled on non-invasive ventilation (either continuous positive airway pressure (CPAP) or bilevel positive airway pressure ventilation (BiPAP)). Moreover, depending on their tolerability, patients were encouraged to self-prone. All patients that deteriorated while on NIV went on to have endotracheal intubation and mechanical ventilation.\n\nEthical approval was obtained as part of the REACT COVID observational study (A longitudinal Cohort Study to facilitate Better understanding and Management of SARS-CoV-2 infection from hospital admission to discharge-across all levels of care): REC Reference 17/NW/0632 SRB Reference Number; SRB0025. Due to the nature of the study, the need for individual informed consent was waived.\n\nData was collected from our electronic clinical information (CIS) system using a combination of semi-automated data extraction and manual collection. We collected baseline demographics (age, gender, duration of symptoms, critical illness severity scores, presence of other comorbidities and laboratory variables), medical treatments received, ventilatory parameters, and position data. Position data are recorded hourly by the ICU nursing staff in the CIS and from this we derived number of prone cycles, timing and total duration. We defined NIV failure as the need for mechanical ventilation. On admission we also collected severity indices such as Acute Physiology and Chronic Health Evaluation (APACHE II), Sequential Organ Failure Assessment (SOFA) scores and oxygenation status. Arterial blood gas (ABG) results were collected alongside patient observations (heart rate (HR) and respiratory rate (RR)) from our CIS to measure change in parameters before, during and after prone position. We assessed the change in oxygenation by measuring the change in the arterial oxygen partial pressure (PaO2 in mmHg) to fractional inspired oxygen (FiO2) ratio (ΔP/F), change in respiratory rate (ΔRR) and heart rate (ΔHR).\n\nAll statistical analysis and data processing were performed using R (R Core Team, Vienna, Austria). Data were tested for normality; those found to be normally distributed were presented as mean and standard deviations (SDs), while non-normally distributed data are presented as median and IQR. Changes in physiological parameters across prone cycles were tested using a paired t-test and are presented and mean and 95% confidence interval. Significance testing between groups was carried out using an independent 2-group t-test for normally distributed variables and the Mann-Whitney U-test for non-normally distributed variables.\n\n\nResults\n\nThere were 81 COVID-19-confirmed patients admitted to the General Intensive Care Unit between 4th of March and 11th of May 2020. The outcomes are up to date as of 26 June 2020. Of those, 20 patients (25%) had a combination of both non-invasive ventilation and self-prone positioning. The mean age of these patients was 53.4 ± 8.3, 55% were male and median admission APACHE II and SOFA severity indices were 11.5 (IQR 5) and 3 (IQR 0) respectively. Among those, 7 patients failed NIV and were subsequently intubated (NIV+IMV group). The characteristics of these patients with severity indices are presented in Table 1. Raw results for each patient are available as Underlying data11.\n\nData are presented in mean ± standard deviation. †Data are presented in median and interquartile range.\n\nAPACHE II, Acute Physiology and Chronic Health Evaluation II score; BMI, body mass index; CPAP, continuous positive airway pressure; CRP, C-reactive protein; eGFR, estimated glomerular filtration rate; EPAP, expiratory positive airway pressure; HS Troponin, High Sensitivity Troponin; INR, International Normalised Ratio; IPAP, inspiratory positive airway pressure; PaO2/FiO2, ratio of arterial oxygen partial pressure to fractional inspired oxygen; SOFA, Sequential Organ Failure Assessment score; WBC, white blood cell count.\n\nThere was a total number of 141 prone cycles performed between these patients. Although the duration of each cycle was variable between cycles and among individual patients, the median duration for each cycle was 3 hours (IQR 2) and the number of proning cycles per patient was 5 (IQR 6.3). Five patients continued with prone positioning beyond 96 hours. Most cycles (46%), were between 1 and 3 hours, a summary detailing prone cycles and duration characteristics is shown in Table 2. Additionally, Figure 1 is a graphical representation of each patient’s time on NIV, demonstrating time spent in prone and supine positions. It follows each of the 20 patients, tracking their position throughout the duration of their admission, up until discharge from ICU or point of intubation.\n\nMedian (Interquartile range).\n\nIMV, Invasive mechanical ventilation; NIV, non-invasive mechanical ventilation.\n\nPatients that failed non-invasive ventilation and required invasive mechanical ventilation (NIV+IMV group) are shown in blue and non-invasive ventilation (NIV) only group in red.\n\nThe outcomes are shown in Table 3. Overall, there was an increment in P/F ratio of 28.7 mmHg (3.83 kPa) (95% CI 18.7-38.6 mmHg, p<0.01) with no change in the heart rate or respiratory rate. The patients who had a successful NIV and prone trial (65%) had a greater increment in P/F ratio of 40.8 mmHg (5.44 kPa) (95% CI 28.8-52.7 mmHg, p<0.01), while those who went on to be intubated did not have a significant improvement in P/F ratio (+5.06 mmHg (0.67 kPa), 95% CI -9.5-19.7 mmHg, p=0.48). Those patients who avoided IMV had a significantly shorter length of hospital stay (11 vs. 28 days, p<0.01) but no other significant differences in outcome or baseline characteristics were observed. Most of the improvement was seen within 24 hours and after this time point the incremental beneficial effect is less for both groups (Figure 2). On average the NIV+IMV group spent 24% of their intensive care unit time pre-intubation in the prone position compared to only 12% for the NIV only group.\n\n*Mean (95% CI). †Median and (Interquartile range). ‡P value <0.05 using Welch’s t-test; §P value <0.05 using Mann-Whitney U-Test.\n\nCI, confidence interval; ICU; intensive care unit; IMV, invasive mechanical ventilation; NIV, non-invasive ventilation; PaO2/FiO2, ratio of arterial oxygen partial pressure to fractional inspired oxygen; SARF, severe acute respiratory failure.\n\nPatients that failed non-invasive ventilation and required invasive mechanical ventilation (NIV+IMV group) are shown as blue dots and non-invasive ventilation (NIV) only group in red dots.\n\nThere were no deaths recorded in either group and all patients who had successful NIV and prone positioning without the need for mechanical ventilation were discharged home. Among those 7 patients who failed NIV (35%), 2 were transferred to the regional extra-corporeal membranous oxygenation (ECMO) centre, both were later discharged home (Table 2). There were no reported cases of any adverse events from these proning episodes.\n\n\nDiscussion\n\nThis is a single-centre retrospective observational study of the clinical outcomes of COVID-19 patients who received a combination of conscious proning and non-invasive ventilation as part of their initial ventilation strategy. Conscious proning and non-invasive ventilation was found to be feasible in 20 patients. Despite variations in patient tolerability and cycle duration, oxygenation improved during prone position period without adverse changes in respiratory rate or heart rate. This response was most marked in patients that did not require escalation to invasive ventilation (65%). Patients that went on to require invasive mechanical ventilation (35%) did not have an improvement in their P/F ratio: modest improvements observed in the first 24 hours (Figure 2) were not sustained beyond this initial period. These patients were similar to patients who received only NIV with respect to APACHE II, SOFA scores and degree of hypoxia defined by P/F ratio on admission.\n\nThe strain that the coronavirus pandemic has placed on national healthcare infrastructure is unprecedented. Although there is only limited data available on the effectiveness of non-invasive ventilation (NIV) in COVID-19, early provision of NIV in moderate to severe acute hypoxic respiratory failure is associated with reduced ICU mortality and intubation rate12. NIV is less resource intensive than IMV and can be managed outside of a critical care environment. NHS England COVID-19 specific guidance suggests that prone position may be of use in NIV patients to improve ventilation/perfusion mismatch, work of breathing and oxygenation13. Whilst the use of prone positioning is well defined and has been extensively evaluated in patients with ARDS by several randomised controlled trials, the benefits of prone positioning in awake, conscious patients with moderate to severe AHRF or ARDS has not been fully explored. Reports from the COVID-19 pandemic from various countries suggests prone positioning in spontaneously ventilating patients may be of value in preventing progression to mechanical ventilation.\n\nOur findings suggest that for a proportion of COVID-19 patients with moderate AHRF (P/F ratio <200 mmHg), non-invasive ventilation in combination with conscious proning can lead to an improvement in oxygenation, less requirement for invasive ventilation and potentially better overall outcomes. All patients with a sustained (>24 hours) positive response to prone positioning avoided intubation, affording them a shorter overall length of hospital stay. However, 35% of patients receiving NIV progressed to invasive mechanical ventilation, despite a similar overall number and duration of proning cycles per patient between both groups. Although none of these patients died, they required a much longer period of hospitalisation and therefore caution is advised when implementing non-invasive ventilation and prone positioning outside of a critical care environment with adequate resources to manage NIV failure.\n\nThe use of prone positioning in COVID-19 pneumonia is supported by our understanding of the pathophysiology of the disease. There is inhomogeneity in the lungs, and CT scans of COVID-19 patients typically shows areas of peripheral ground glass changes that later develop into linear consolidations14. Areas of exudation, macrophage infiltration, fibrosis and mucous plugs are typical findings on autopsy of deceased patients with COVID 1915. Placing patients in the prone position may help to drain secretions from the lung peripheries, improve lung dyshomogeneity, recruitment and ventilation/perfusion mismatch. Many international guidelines recommend prone positioning for intubated and mechanically ventilated patients for these reasons, so it seems reasonable to conclude that similar benefits may be gained by prone positioning in non-invasively ventilated patients with similar underlying pathology.\n\nThere are obvious risks associated with treating patients with AHRF with non-invasive ventilation. The primary risk to the patient is inappropriate delay in intubation and ventilation. COVID-19 AHRF frequently meets the criteria for ARDS and previous studies have suggested that in ARDS, delaying intubation due to the use of NIV is associated with increased mortality16. Likewise, the SARS-CoV-2 virus is transmitted by respiratory droplets; non-invasive ventilation has been shown to produce droplets of >10 μm in size that are largely deposited on surfaces within a 1 meter radius17. Therefore, NIV could potentially increase the risk of virus transmission to individuals in close proximity with the patient. This needs to be a consideration when devising guidelines for personal protective equipment and appropriate cohort allocation of infected patients to minimise infection risk.\n\nThere are some limitations of this study. This is a single-centre, retrospective cohort study only limited to small number of COVID-19 patients admitted to the intensive care unit setting with the option of subsequent escalation of care to mechanical ventilation. Most patients were in general young, with a median age of <60 years old and able to self-prone with non-invasive ventilation. These findings may not be transferable to older patient’s group or patients with severe acute hypoxic respiratory failure with P/F ratio of <100mmHg. The study was observational, there were no set criteria for NIV proning and NIV failure with subsequent endotracheal intubation. The clinical judgement and subsequent interventions provided may have been variable between individual senior clinicians and may not be reflective of other centres. Additionally, position data are only recorded hourly and as such some granularity of the data may be lost when the cycles were much shorter period of less than an hour.\n\nDespite these limitations, our results are in line with the other recently published studies of conscious prone positioning in COVID-19 pneumonia. Several case reports and small (up to 25 patients) observational studies conducted in multiple settings (outside ICU, emergency department) with variations in respiratory support (non-invasive ventilation/high-flow nasal oxygen/standard face mask oxygen therapy) and varying severity of hypoxemia has demonstrated beneficial effects of prone positioning in COVID-19 pneumonia. All these studies suggest conscious prone position is associated with an increment in oxygenation and recovery without the need for mechanical ventilation in most cases8–10. Our results demonstrate, specifically that a sustained response across multiple cycles, for a period >24 hours, is associated with successful treatment with NIV. Taken together, these results indicate that prone positioning in awake, non-intubated patients, in combination with non-invasive ventilation is feasible and may be considered as an early intervention in COVID-19 respiratory failure, particularly in the context of a severe pandemic to prevent mechanical ventilation and its subsequent complications. They also suggest that a loss of response to prone position may potentially be a sign of NIV failure and warrant early evaluation and consideration for endotracheal intubation.\n\n\nConclusion\n\nIn conclusion we demonstrate that prone positioning in conjunction with NIV can improve oxygenation in patients with COVID-19. This can be achieved without significant adverse effects and particularly in those with a sustained response, may avoid intubation. When used in a suitably monitored environment, with access to experienced clinicians able to facilitate invasive mechanical ventilation if required, prone positioning alongside NIV may be a useful tool in treating COVID-19 patients with moderate acute hypoxic respiratory failure.\n\n\nData availability\n\nFigshare: Conscious proning _Burton-Papp 2020.numbers. https://doi.org/10.6084/m9.figshare.12676565.v211.\n\nThis project contains de-identified data for each patient, including details of proning and type of non-invasive ventilation received.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Author information\n\nThe University Hospital Southampton Critical Care Team comprise Dr Sanjay Gupta, Dr Julian Nixon, Professor Michael P W Grocott, Professor Denny ZH Levett, Dr Michael Stewart, Dr Ahilanadan Dushianthan, Dr David Sparkes, Dr Robert Chambers, Dr Kathleen Nolan, Dr Suzie Tanser, Dr Jonathan Fennell, Dr Michael Celinski, Dr Dominic Richardson, Dr Rebecca Cusack, Dr Benjamin Skinner, Dr Timothy Nicholson-Robert, Dr Mai Wakatsuki, Dr Ben Thomas and Dr Francois Wessels.\n\nThe REACT COVID Investigators comprise Professor Tom Wilkinson, Dr Anna Freeman, Dr Hannah Burke, Dr Ahilanadan Dushianthan, Dr Michael Celinski, Professor James Batchelor, Professor Saul Faust, Professor Gareth Thomas and Professor Christopher Kipps.\n\n\nReferences\n\nCheung TMT, Yam LYC, So LKY, et al.: Effectiveness of noninvasive positive pressure ventilation in the treatment of acute respiratory failure in severe acute respiratory syndrome. Chest. 2004; 126(3): 845–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSivaloganathan A, Nasim-Mohi M, Brown MM, et al.: Noninvasive ventilation for COVID-19 associated acute hypoxaemic respiratory failure: experience from a single centre. Br J Anaesth. 2020. Publisher Full Text \| Free Full Text\n\nRoberts CM, Levi M, McKee M, et al.: COVID-19: a complex multi-system disorder. Br J Anaesth. 2020. Publisher Full Text \| Free Full Text\n\nBloomfield R, Noble DW, Sudlow A: Prone position for acute respiratory failure in adults. Cochrane Database Syst Rev. 2015; 2015(11): CD008095. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuérin C, Reignier J, Richard JC, et al.: Prone positioning in severe acute respiratory distress syndrome. N Engl J Med. 2013; 368(23): 2159–68. PubMed Abstract \| Publisher Full Text\n\nAlhazzani W, Møller MH, Arabi YM, et al.: Surviving Sepsis Campaign: guidelines on the management of critically ill adults with Coronavirus Disease 2019 (COVID-19). Intensive Care Med. 2020; 46(5): 854–887. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThe Australian and New Zealand Intensive Care Society (ANZICS) COVID-19 Guidelines Version 1. Melbourne, 2020. Reference Source\n\nElharrar X, Trigui Y, Dols AM, et al.: Use of Prone Positioning in Nonintubated Patients With COVID-19 and Hypoxemic Acute Respiratory Failure. JAMA. 2020; 323(22): 2336–2338. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSartini C, Tresoldi M, Scarpellini P, et al.: Respiratory Parameters in Patients With COVID-19 After Using Noninvasive Ventilation in the Prone Position Outside the Intensive Care Unit. JAMA. 2020; 323(22): 2338–2340. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChad T, Sampson C: Prone positioning in conscious patients on medical wards: A review of the evidence and its relevance to patients with COVID-19 infection. Clin Med (Lond). 2020; 20(4): e97–e103. PubMed Abstract \| Publisher Full Text\n\nDushianthan A, Burton-Papp H, Jackson, A: Conscious proning _Burton-Papp 2020.numbers. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12676565.v2\n\nLiu YJ, Zhao J, Tang H: Non-invasive ventilation in acute respiratory failure: A meta-analysis. Clin Med (Lond). 2016; 16(6): 514–23. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSpecialty Guides for Patient Management during the Coronavirus Pandemic. NHS England. 2020. Reference Source\n\nHani C, Trieu NH, Saab I, et al.: COVID-19 pneumonia: A review of typical CT findings and differential diagnosis. Diagn Interv Imaging. 2020; 101(5): 263–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQaqian M, Lin W, Wen J, et al.: Clinical and pathological characteristics of 2019 novel coronavirus disease (COVID-19): a systematic review. medRxiv. 2020. Publisher Full Text\n\nAntonelli M, Conti G, Esquinas A, et al.: A multiple-center survey on the use in clinical practice of noninvasive ventilation as a first-line intervention for acute respiratory distress syndrome. Crit Care Med. 2007; 35(1): 18–25. PubMed Abstract \| Publisher Full Text\n\nSimonds AK, Hanak A, Chatwin M, et al.: Evaluation of droplet dispersion during non-invasive ventilation, oxygen therapy, nebuliser treatment and chest physiotherapy in clinical practice: Implications for management of pandemic influenza and other airborne infections. Health Technol Assess. 2010; 14(46): 131–72. PubMed Abstract \| Publisher Full Text" }	[ { "id": "68510", "date": "04 Sep 2020", "name": "John Whittle", "expertise": [ "Reviewer Expertise Critical Care and Perioperative 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\nIn this retrospective observational cohort study, 20 spontaneously ventilating patients admitted to critical care with hypoxic respiratory failure consequent to COVID 19 pneumonia, who underwent proning while receiving non-invasive ventilation were identified from the EHR. The primary outcome examined was a change in PaO2/FiO2 (P/F) ratio while prone as compared with supine, which the authors report as improved while prone.\nIn terms of data quality and study design, since this is a retrospective study, no single protocol for awake proning was used, creating a large amount of variability in duration, cycle length and frequency of intervention. Similarly, total length of time spent on NIV, and total number of hours /days spent awake proning appears very varied. This reduces the utility of the data significantly. No comparative statistics are presented between groups (correctly).\n\nIn terms of trajectory of illness, it would be helpful to plot the overall P/F ratio over time for both intubated and non intubated groups, since from the data available it would appear that those patients who were ultimately intubated started with worse oxygenation and benefited least from the intervention.\nIt may also be useful to examine a control group of patients who did not self-prone, but did receive non-invasive ventilation during the study period.\nThe data presented do however, provide reassurance in terms of safety of the intervention in terms of hemodynamic and respiratory stability.\nI do not agree that the data presented in this study demonstrate the potential for better overall outcomes with awake proning in COVID-19. More data is required to demonstrate that the trajectory of illness was altered by proning, which is not available in this study. Rather, the observation that patients who avoided intubation overall spent lower percentage of time prone and had a increased response to proning in terms of P/F ratio, suggests that those patients on the trajectory (who are not well described in this study) towards intubation did not have their outcome altered by proning. Rightly, in the discussion the authors discuss the risks associated with applying awake proning.\nIn summary Burton-Papp et al. have shown that P/F ratio may improve in proned patients on non-invasive ventilation with COVID-19 respiratory failure. Their data also suggests that awake proning on NIV in COVID-19 positive patients is safe in terms of cardiorespiratory stability in those patients who are able to tolerate the intervention. This intervention may therefore provide some clinical benefit, but it is not possible to determine from this study with limited numbers where no control group was presented. What they have not demonstrated is whether proning on NIV alters clinical course, prevents intubation, what protocol is most effective or who might benefit most from this intervention all of which would require prospective study or matched controlled. They further did not describe which features prevented patients from tolerating awake proning on NIV which may be useful in understanding the impact of the intervention.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "72632", "date": "21 Oct 2020", "name": "Ivan Pavlov", "expertise": [ "Reviewer Expertise Emergency medicine", "respiratory support with HFNC", "awake proning." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 retrospective report on a subgroup of 20 patients with severe COVID-19 who were able to self-prone while receiving non-invasive ventilation in the ICU, among a grand total of 81 patients admitted to the ICU during the study period.\n\nThe primary outcome reported by the authors is change in PaO2/FiO2 ratio while in prone position. Clinical outcomes, such as mortality and the incidence of intubation, are also reported.\n\nThe retrospective nature of the report precludes any causal inferences with regards to the efficacy of awake proning. However, the authors do show that intubation can indeed be avoided in some patients with moderate ARDS due to COVID-19, and that awake proning is associated with improvements in PaO2/FiO2 ratios.\n\nThe authors assert that improvement in PaO2/FiO2 ratios predicted avoidance of intubation. Their data do not allow us to conclude whether this improvement is solely the response to awake proning, or natural evolution of patients who were already getting better in the first place, and would have avoided intubation whether or not they were subjected to awake proning. One may also wonder if reverse causality was also at play, and if patients who didn't improve their PaO2/FiO2 ratio were intubated precisely for that reason. Intubation is a soft outcome that relies on treating physician judgement, and it is difficult to disentangle cause and effect in this scenario.\n\nIn summary, the authors have shown that PaO2/FiO2 ratios may improve in response to awake proning, and they did not identify any obvious safety signals in this cohort of patients that were able to tolerate awake self-proning. Although this paper does not demonstrate efficacy of awake proning, it does add to the growing body of knowledge on this subject and allows comparisons with other cohorts which used different strategies in terms of duration of proning, as well as the type of respiratory support. Publication of such prelimary data is important, especially while awaiting definitive answers from randomized controlled trials.\n\nIs the work 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/9-859
https://f1000research.com/articles/9-857/v1	31 Jul 20	{ "type": "Systematic Review", "title": "Does health and social care provision for the community dwelling older population help to reduce unplanned secondary care, support timely discharge and improve patient well-being? A mixed method meta-review of systematic reviews", "authors": [ "Shoba Dawson", "Patience Kunonga", "Fiona Beyer", "Gemma Spiers", "Matthew Booker", "Ruth McDonald", "Ailsa Cameron", "Dawn Craig", "Barbara Hanratty", "Chris Salisbury", "Alyson Huntley", "Shoba Dawson", "Patience Kunonga", "Fiona Beyer", "Gemma Spiers", "Matthew Booker", "Ruth McDonald", "Ailsa Cameron", "Dawn Craig", "Barbara Hanratty", "Chris Salisbury" ], "abstract": "Background: This study aimed to identify and examine systematic review evidence of health and social care interventions for the community-dwelling older population regarding unplanned hospital admissions, timely hospital discharge and patient well-being. Methods: A meta-review was conducted using Joanna Briggs and PRISMA guidance. A search strategy was developed: eight bibliographic medical and social science databases were searched, and references of included studies checked. Searches were restricted to OECD countries and to systematic reviews published between January 2013–March 2018. Data extraction and quality appraisal was undertaken by one reviewer with a random sample screened independently by two others. Results: Searches retrieved 21,233 records; using data mining techniques, we identified 8,720 reviews. Following title and abstract and full-paper screening, 71 systematic reviews were included: 62 quantitative, seven qualitative and two mixed methods reviews. There were 52 reviews concerned with healthcare interventions and 19 reviews concerned with social care interventions. This meta-review summarises the evidence and evidence gaps of nine broad types of health and social care interventions. It scrutinises the presence of research in combined health and social care provision, finding it lacking in both definition and detail given. This meta-review debates the overlap of some of the person-centred support provided by community health and social care provision. Research recommendations have been generated by this process for both primary and secondary research. Finally, it proposes that research recommendations can be delivered on an ongoing basis if meta-reviews are conducted as living systematic reviews. Conclusions: This meta-review provides evidence of the effect of health and social care interventions for the community-dwelling older population and identification of evidence gaps. It highlights the lack of evidence for combined health and social care interventions and for the impact of social care interventions on health care outcomes. Registration: PROSPERO ID CRD42018087534; registered on 15 March 2018.", "keywords": [ "meta-review", "systematic reviews", "health care", "social care", "community-dwelling older population", "unplanned admissions", "patient well-being" ], "content": "Introduction\n\nIn a recent government report on the UK population, it is predicted that in 50 years’ time there will be an extra 8.2 million people aged 65 years and above in the UK. This cohort will comprise over a quarter of the total UK population and equates to the current size of London1. We are already aware of the impact of the ageing population on health care services. In 2017, 3.5 million (22.2%) of total hospital admissions in England were in the 75 years and over age group. Once in hospital, the same population spent 10.7 million days as inpatients if they were not in the last year of life and 7.5 million days if they were in their last year of life2. Once in hospital, there are further issues that need to be considered. Longer, potentially unnecessary hospital stays (delayed discharge) are likely to have a detrimental effect on the older population. NHS Improvement reports that 35% of 70-year-old inpatients experience a decline in function compared to preadmission, and this rises to 65% for people over the age of 903.\n\nDischarge care for the older population often involves ongoing health care and may also involve social care provision. Social care provision is a balance for local authorities between protecting the most vulnerable in society and the resources available. Insufficient numbers of care home staff and affordable care home places can result in older people having an inappropriately extended hospital stay4. The National Audit office estimate that the number of older inpatients no longer requiring acute care but still in hospital is around 2.7 timers higher than official statistics suggest5. The lack of integration of the health and social care sectors is historical as well as financial and how to improve the situation has been a challenging and controversial debate for decades. In 2014, The Barker Commission concluded that the support needed by older people to navigate through the existing health and social care system needed to be simplified, with services built around people’s individual needs. It further concluded that integration of health and social care provision can only happen if traditional definitions and divides are broken down6.\n\nIn 2017, NHS England set out an ambition to make a national, comprehensive move to integrated health and social care by the development of sustainability and transformation partnerships (STPs)7. These STPs are local partnerships of NHS and local authority organisations with the aim of taking more control of local funding and services to improve the health and wellbeing of the population8.\n\nThe overall aim of this meta-review is to provide an evidence base of efficacy for health care, social care and combined health and social care interventions for the older population in terms of the impact on hospital admissions, timely discharge and patients’ quality of life.\n\nThe aims of this meta-review were:\n\nA) To identify effective interventions to deliver health and social care to the community-dwelling older population.\n\nB) To understand what is important to the community dwelling older population concerning their care, and important to the professionals providing it, with respect to unplanned hospital admissions, inpatient stays and patient wellbeing.\n\nC) To identify definitions of social care and combined health and social care for older people that have been used in the systematic review literature.\n\nD) To identify the components of the health and social care interventions that potentially complement and reduce unplanned hospital admissions, support timely appropriate hospital discharge and enhance well-being.\n\nE) To identify future mixed-methods synthesis by matching intervention effectiveness with related patient experience to facilitate suggestions for effectiveness or ineffectiveness of intervention approaches and produce research recommendations.\n\n\nMethods\n\nWe were guided by the Joanna Briggs methodology for Joanna Briggs Institute umbrella reviews9 and reported according to PRISMA guidance10. A search strategy was developed in collaboration with the research team with the advice of an experienced information specialist (FB). It combined the following concepts: ([Health care terms or social care terms] AND [overview of reviews filter])11 NOT [LMIC (lower- and middle-income countries) OR children] (Extended data, Appendix 112). It is more robust to exclude LMIC countries than to select studies where higher income countries are indexed, since indexing terms for higher income countries are not always added. The search did not specify ‘older people’ because scoping revealed that not all relevant studies were indexed in this way.\n\nThesaurus headings for each concept were combined with terms in the title and abstract fields and translated as appropriate for each database. All search results were downloaded to Endnote and de-duplicated. The following databases were searched: The Cochrane Library (Wiley); MEDLINE (OVID); Embase (OVID); Cumulative Index to Nursing and Allied Health Literature (CINAHL) (EBSCO); PsycINFO (OVID); Social Sciences Citation Index (Web of Knowledge); Conference Proceedings Citation Index - Social Science & Humanities (Web of Knowledge); International Bibliography of the Social Sciences (IBSS) (ProQuest); Social Services Abstracts (ProQuest); Social Care Online.\n\nSearches were restricted to Organisation for Economic Co-operation and Development (OECD) countries and to the past five years (January 2013– March 2018). A 2013 limit ensured capturing data from at least previous 30–40 years, as determined by examining relevant 2009/2013 systematic reviews for their search limits. Thus, any studies on recent changes to care provision such as the GP Contract Changes of 2004, the introduction of four-hour wait targets in emergency departments in 2004 and the Health & Social Care Act 2008 were included. There were no language restrictions, provided an English language abstract was available for initial screening. Forward and backward referencing was conducted on all included full systematic review papers to identify any further relevant systematic reviews. Detailed methods can be found in our PROSPERO registration protocol CRD42018087534.\n\nWe sought the highest-level evidence available for our research questions. In our health care question, we expected that to be systematic reviews of randomised controlled and controlled trials. In our social care and combined health and social care questions we expected systematic reviews of controlled/observational studies. We used the DARE guidance to assess whether a review can be classified as a systematic review13.\n\nCommunity-dwelling older (≥65 years) people. If systematic reviews included data from participants older and younger than 65 years of age, we only included the systematic reviews if the older adult studies were presented separately and formed at least 50% of the included studies. Community dwelling was defined as all residential living including domestic, care, and nursing and sheltered (extra care) housing. Reviews that solely focus on carers or patients in end-of-life care were excluded, as this is beyond the scope of the current review.\n\nHealth care interventions were those received by a community-dwelling older population that did not involve include an admission into a secondary or tertiary care hospital.\n\nSocial care: as defined in the Care Act 2014:\n\n\"Adult social care\" -\n\na) includes all forms of personal care and other practical assistance for individuals who by reasons of age, illness, disability, pregnancy, childbirth, dependence on alcohol or drugs or any similar circumstances, are in need of such care or other assistance, but\n\nb) does not include anything provided by an establishment or agency for which Her Majesty’s Chief Inspector of Education, Children’s Services and Skills is the registration authority under section 5 of the Care Standards Act of 2000\"14.\n\nCombined health and social care: any combination of the above, whether provided by separate services but in a co-ordinated way, or through fully integrated services.\n\nFor the quantitative reviews, any comparator was suitable in the studies described in the included systematic reviews. For the qualitative reviews this was not applicable.\n\nOutcomes for quantitative systematic reviews were: 1) unplanned hospital admissions/readmissions, 2) length of stay (LOS) and 3) patient well-being.\n\nWe were inclusive in our use of patient well-being outcomes and, in addition to measures of the quality of life, we included measures of social isolation and loneliness. We included systematic reviews of qualitative data if they described: patients’ or health and social care professionals’ experiences of healthcare or social care or combined health and social care relevant to unplanned hospital (re)admission or timely discharge and/ or quality of life.\n\nTitles and abstracts were independently assessed for inclusion by two reviewers using pre-defined criteria as detailed above. Disagreements were resolved through discussion and, where necessary, consultation with a third reviewer. For publications of potential relevance, full papers were assessed independently by two reviewers with disagreements resolved as above. Data extraction was undertake using a customised spreadsheet (Extended data, Appendix 212) in Microsoft Excel by one reviewer (SD) with a random sample of 20% independently screened by two other authors.\n\nWe were guided by the Joanna Briggs methodology for umbrella reviews9. All data analysis was predominantly descriptive in nature. Where data synthesis was performed it comprised of narrative groupings of data or ideas.\n\nDescriptive analysis of evidence\n\nWe narratively presented the aims, specific intervention definition, outcome measured and authors conclusions from both quantitative and qualitative reviews in both text and tables grouped by overall intervention type. If there were more than one systematic review of the same or similar data, we have reported any differences.\n\nRisk of bias (quality) assessment\n\nQuality appraisal of the systematic reviews of quantitative data was conducted using the ROBIS tool15. This comprises four main domains assessed by signalling questions: eligibility, identification of studies, collection/appraisal of data, and synthesis and findings. Quality appraisal of systematic reviews of qualitative data and mixed methods reviews was conducted using the GRADE-CERQual tool16. This approach includes four components for assessing how much confidence to place on the findings: the methodological limitations of the individual qualitative studies contributing to the review finding; the relevance to the review question of the individual studies contributing to the review finding; the coherence of the review finding; and the adequacy of data supporting a review finding.\n\nQuality appraisal was conducted independently by two reviewers for a 10% sample of included systematic reviews. There was a high level of agreement between the reviewers (94%), therefore the remaining reviews were appraised by one reviewer (SD) and checked by a second reviewer (AH). Any discrepancies were resolved through consensus. We did not exclude on quality but took account of the quality of evidence when discussing the findings of the included systematic reviews.\n\nEvidence map (addressing Aims A & B)\n\nAn evidence map was produced detailing volume of evidence (number of reviews) per intervention per outcome measure and showing gaps in evidence.\n\nEvidence summary (addressing Aims A & B)\n\nAn evidence summary was produced for interventions which had a positive or no impact on our outcomes of interest. Positive or no impact were defined as any stated by the authors’ results and conclusions at systematic review level. The majority of these were based on evidence from A) meta-analysis of randomised controlled trial data; B) narrative evidence predominantly from RCT data, but no meta-analysis performed, C) limited evidence means two or less RCTs, D) low quality evidence means predominantly non-RCT evidence. A small number of qualitative reviews were identified and where present are denoted by Q.\n\nDefinitions of combined health and social care (addressing Aim C)\n\nIf present these were collated in one table and discussed within the results and discussion section.\n\nComplementary components of health and social care interventions (Addressing Aim D).\n\nThese were described in the results and the discussion sections.\n\nFuture mixed methods synthesis (addressing Aim E).\n\nWe identified existing and future mixed methods synthesis of evidence to facilitate suggestions for future research.\n\n\nResults\n\nThere were 71 systematic reviews included in this meta-review: 62 quantitative reviews, seven qualitative reviews and two mixed methods reviews (Extended data, Appendix 3: PRISMA diagram12) 52 reviews were concerned with health care interventions, of which 46 were quantitative reviews and six were qualitative reviews. 19 reviews were concerned with social care interventions of which 16 were quantitative, two were mixed methods and one qualitative review. Extended data, Table 112 contains a description of the included reviews.\n\nPatient populations. Systematic reviews of health care interventions focused on the older population (17), chronic obstructive pulmonary disease (COPD) (12), heart failure/atrial fibrillation (10) with a smaller number of reviews on stroke (2), dementia (1), Parkinson’s (1), vertebral compression fractures (1) and mixed chronic conditions (2). In addition, there were six composite reviews covering broader topic areas. Systematic reviews of social care included the older population (16) and dementia (3).\n\nTypes of interventions. Health care interventions were organised into three groups: Care in the community (Reviews 1–35)17–51, Urgent care at the community/hospital interface (Reviews 36–39)52–55 and Discharge and transitional care at the hospital/community interface (Reviews 2, 40–52)18,56–68. Social care interventions were organised into two groups: formal social care (Reviews 53–57)69–73 and synthetic social support (Reviews 58–71)74–87.\n\nHealth care interventions. Of the 46 quantitative reviews of health care interventions, 28 (61%) were determined to be at low risk of bias across all four domains, 13 (28%) had a least one domain determined to be unclear (due to lack of information), and five (11%) had at least one domain (predominantly domains 2 and 3) determined to be at high risk (see Extended data, Table 2a12).\n\nThere were six qualitative reviews concerned with health care interventions and of these, five were determined to be at low concern across all four domains and high confidence overall. The remaining one review was determined to be of moderate concern over domains two and three and therefore of moderate confidence.\n\nSocial care interventions. Of the 16 quantitative reviews of social care interventions, eight (50%) were determined to be at low risk of bias across all four domains, four (25%) had one domain determined to be unclear (mostly domain 3) and four (25%) were determined to have at least one domain at high risk (mostly domain 3 and 4) (see Extended data, Table 2b12).\n\nThere were one qualitative and two mixed methods reviews concerned with social care interventions and of these, one was determined to be at low concern across all four domains and high confidence. The other review was determined to be a mixture of moderate and low concern and of moderate confidence.\n\nIn this section, the following are summarised: 1) evidence and evidence gaps for health and social care interventions per condition and outcome, and 2) the effectiveness of interventions and people’s experiences of them. Finally, detailed descriptions of the included systematic reviews are given.\n\nThe majority of included health care systematic reviews measured hospital admissions as an outcome (33/52). Two formal social care reviews measured hospital admissions (2/5). No synthetic social support reviews measured hospital admissions (0/14) (Extended data, Table 3 shows an evidence map12).\n\nTimely discharge was only measured in the discharge care intervention reviews (2/14) and was not present in any social care review (0/19).\n\nWithin health care systematic reviews, quality of life was measured in 20/52 reviews and was most prevalent in the care in the community intervention reviews, particularly self-management, exercise/rehabilitation and medication review (17/35), as opposed to 0/4 in the urgent care reviews and 3/14 in the discharge care reviews. Quality of life was measured in 17/19 social care intervention reviews of which 10 were focused on social isolation and loneliness.\n\nOnly 7/71 of the included systematic reviews were of qualitative data (people’s experiences) and 2/71 contained a combination of quantitative and qualitative data with similar representation across health (6/52) and social care (3/19) reviews.\n\nPositive benefit. The evidence for positive benefit of interventions in reducing hospital admissions is derived from the health care evidence across community, urgent and discharge care interventions focusing on the older population, COPD and heart failure patients (Extended data, Table 4 provides a summary of the evidence12).\n\nIn the older population there is meta-analysis-level evidence for positive benefit for both discharge/transitional care for all and influenza vaccination for nursing home residents. For COPD patients there is meta-analysis level evidence for positive benefit for rehabilitation/post-rehabilitation support, influenza vaccination, discharge/transitional care and hospital at home (in place of admission). For heart failure patients there is meta-analysis level evidence for positive benefit for discharge/ transitional care and hospital-initiated case management.\n\nNo benefit. There is meta-analysis level evidence for no benefit in terms of hospital admissions for the following health interventions. For the general population, community case management, medication review and nurse-led geriatric ED care confers no benefit. For COPD patients, self-management intervention confers no benefit and for heart failure patients supervised exercise and community case management confers no benefit.\n\nPositive benefit. There is meta-analysis level evidence for positive benefit of hospital-initiated case management for heart failure patients.\n\nNo benefit. There is no meta-analysis level evidence for any health or social care intervention in any population supporting no benefit for timely discharge.\n\nPositive benefit. The evidence for positive benefit of interventions in quality of life is present in both health and social care interventions with meta-analysis level evidence for the older population, COPD, heart failure, stroke and dementia patients. For the older population, this includes self-management and reablement interventions; for COPD patients, this includes breathing techniques, Tai Chi and hospital at home (in place of admission) interventions; for heart failure patients, this includes general exercise, Tai Chi, hospital at home (in place of admission), discharge and transitional care; for stroke patients this includes self-management.\n\nNo benefit. There is meta-analysis level evidence that supports no benefit for quality of life for the older population with medication review and for COPD patients with post rehabilitation support.\n\nThere are nine included systematic reviews of qualitative evidence or a combination of quantitative and qualitative data (mixed methods) evidence: self-management and case management for heart failure patients, self-management for stroke patients, transitional and discharge interventions for older people, rehabilitation and exercise for COPD patients, formal social care (mixed methods) for the older population and reablement (mixed methods) for the older population, These are described in Extended data, Table 112) and discussed in more detailed in aim E.\n\nDefinitions. Definitions of specific social care interventions are listed in Extended data, Appendix 412 and include reablement, personal support and synthetic social support.\n\nDefinitions of combined health and social care were poorly described in the included systematic reviews (Extended data, Appendix 412). All 13 definitions identified came from the health care systematic reviews with eight describing care in the community interventions and five describing discharge/transitional interventions. Combined health and social activity were implied as opposed to specifically mentioning integrated working in all these definitions with the exception of Review 4157. This review included discharge and transitional interventions defined as ‘interventions that could be implemented in any health or social care setting (primary, secondary or community care), as long as they crossed the boundary between two or more settings. The community setting encompassed care given in the community, in patient homes or by social care professionals.’\n\nComplementary components of health and social care interventions. Aim D was to identify complementary components of care across health and social care interventions. By examining the definitions of individual health and social care interventions it is clear to see some overlap of components for the social care interventions reablement and personal assistance with aspects of health professional-driven interventions of self -management, case management, discharge/transitional care and rehabilitation (Extended data, Appendix 412). These interventions included a home-based, tailored/individualized (patient-centered) approach, one to one, face to face, co-ordination of integrated care to support people to live their lives as well as possible for as long as needed.\n\nMixed methods synthesis that matched evidence of intervention effectiveness with related patient experience evidence were identified (Extended data, Table 512). This was limited because only seven systematic reviews with qualitative data25,27,29,43,61,62,70 and two mixed methods reviews78,85 were included in this meta review.\n\nTwo systematic reviews of qualitative evidence were conducted as part of segregated mixed methods reviews. Stroke and self-management (Reviews 10 & 11)26,27 was conducted by the same authors as was heart failure and case management (Reviews 26 & 27)42,43. In the latter case, the qualitative synthesis sought to understand the impact of case management on hospital admissions42 for patients with heart failure in a qualitative synthesis43.\n\nTwo of the systematic reviews were conducted as integrated mixed methods reviews examining social care for the older person and reablement for the older person69,71. Social care for the older person is also evaluated in one systematic review of qualitative data (Review 54)70 as is reablement with a systematic review of quantitative data (Review 56)72.\n\nThere are two future mixed methods synthesis identified by the meta-review that could be conducted: 1) Rehabilitation and post rehabilitation support for COPD patients and 2) Discharge/transitional care for the older population 3) Rehabilitation and post rehabilitation support for patients with COPD is evaluated in three effectiveness systematic reviews (Reviews 12, 14, 15)28,30,31 with a complementary systematic review of qualitative data in Review 1329. Discharge and transitional care for the older person is evaluated in four effectiveness reviews (Reviews 40-43)56–59 with two complementary systematic reviews of qualitative data in Reviews 45 and 4661,62.\n\n\nDiscussion\n\nThis meta-review describes and evaluates health and social care provision aiming to reduce unplanned secondary care, support timely discharge, and improve patient well-being for the community dwelling older population.\n\nThere were 71 systematic reviews included: 62 quantitative reviews, seven qualitative reviews and two mixed methods reviews. Of these, 52 reviews were concerned with health care interventions, and 19 reviews were concerned with social care interventions. There was very little content of these included reviews providing evidence for combined health and social care for the older person which reflects traditional mindsets in practice and research both in the UK and elsewhere8. The reviews included health care interventions targeting the older population as well as specific patient populations such as COPD, heart failure and dementia patients. The reviews of social care interventions predominantly included the older population with some of the studies within the reviews, including younger populations. There were fewer reviews of social care than health care, and within these reviews, there were fewer RCTs than in the health care reviews. Quality appraisal of the health and social care reviews shows that a greater proportion of the health care reviews compared to social care reviews were of a higher methodological standard. This was expected, as systematic review methodology is less common in social care research88. However, it is of note that 50% of the social care reviews were determined to be of low risk of bias overall.\n\nThis meta-review maps out intervention research by population and outcome, highlighting evidence but also identifies evidence gaps. Some of these evidence gaps are not of value to pursue; for example, we would not expect most community and urgent care interventions to improve timely discharge. It is unlikely that synthetic social support would impact on hospital admissions.\n\nHowever, there is an interesting dichotomy in intervention research and measurement in quality of life outcomes; whilst it is a widespread and obvious outcome for self-management, exercise and rehabilitation interventions, it is less prevalent in more medical care provision such as case management, urgent care, discharge and transitional interventions. Patient involvement and satisfaction with care is not only important for patients’ quality of life but also can impact the success of an intervention89. The qualitative data exploring patient and health professional experience when available, can also provide unique insight regarding a specific intervention90.\n\nSocial care and synthetic social support evidence in this meta-review tends to focus on subjective outcomes and yet it could be of value for social care provision research to measure more objective outcomes such as the use of care services. The evidence for effectiveness in this meta-review is dominated by hospital admissions and quality of life. Timely discharge is more likely to be the focus of hospital-based care. Across the 71 included systematic reviews, we only identified seven relevant reviews of qualitative evidence and two reviews of mixed-methods evidence. Those interventions measuring hospital admissions were health care focused, and the included systematic reviews were generally supported by a meta-analysis of RCTs. Quality of life outcomes was present in both health and social care systematic reviews; notably, there was no meta-analysis level evidence in social care or synthetic social support interventions reviews. Social care data comprised few RCTs in combination with less rigorous study types of a heterogeneous group of interventions.\n\nA total of 13 reviews focused on combined or integrated health and social care interventions, with only one systematic review having a clear, integrated health and social care definition for its included studies57. Despite the fact we know that health care works with social care within interventions described in the many of the included systematic reviews of health interventions, e.g. ED, discharge and transitional care interventions, there is little mention of social care involvement in these health research publications91. Although we are looking at systematic review-level evidence, this likely reflects the individual study intervention descriptions. This reinforces the view that we persist in researching health care and social care as separate entities and are not acknowledging the combined activities that already exist. Hopefully, we are on the cusp of change with combined health and social care funding coming in to place92. It is also important to acknowledge that more pragmatic evaluation work of health and social care at a local level may be addressing this better.\n\nFinally, we identified mixed methods evidence synthesis within the meta-review. This aim had two objectives. Firstly, to demonstrate the utility of mixed method synthesis in this topic area. For example, the evidence for case management for heart failure both examines effectiveness data for potential reduction of hospital admissions as well as qualitative data exploring patients’ (and health professionals’) experiences of case management42,43. Secondly, to identify future mixed-method synthesis, i.e. research recommendations for future work. Several topic areas were identified across both health and social care (Extended data, Table 512) with notable examples being the use of quantitative (effectiveness) and qualitative (patient experience) in discharge and transitional care56–59,61,62.\n\nThere are limitations to conducting a meta-review on such a broad topic area. This meta-review included systematic reviews published in the past five years. This means if a topic area had been reviewed with a definitive outcome prior to this, the evidence does not appear in this meta-review. Some interventions may not have enough primary RCTs or studies generally to warrant a systematic review, e.g. diuretic management of heart failure patients in the community.\n\nA meta-review can only really express a direction of effect of evidence for an intervention type, and the detail is needed to get the full picture. An example of this is the evidence for nurses and geriatricians working in emergency departments. At meta-review level, the evidence suggests that nurses do not impact on patient admission, but geriatricians do. At the study level, we can see that the nurse studies are of high quality and component analysis suggests that the impact depends on the type of nurse and the context53. The geriatrician data comprises lower quality observational studies and therefore needs replication with RCTs55.\n\nThe searches for this meta-review were conducted in 2018, reflecting the length of time needed to synthesise such a large volume of evidence. The fact that the meta-review has included systematic reviews and not primary studies give us confidence that the overall evidence base of the interventions is unlikely to have significantly changed. However, this topic area is an excellent candidate for becoming a living systematic review to continue to inform future primary and secondary studies92,93.\n\nIn conclusion, this meta-review of health and social care interventions for the older population provides evidence about the overall effect and the evidence gaps of the included interventions, with qualitative data for selected topics. It also highlights the lack of evidence, detail and discussion for combined health and social care and the lack of high-quality evidence for the impact of social care interventions on care provision outcomes. This meta-review will support future research in health and social care in the ageing population.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: Does health and social care provision for the community dwelling older population help to reduce unplanned secondary care, support timely discharge and improve patient well-being? A mixed method meta-review of systematic reviews. https://doi.org/10.6084/m9.figshare.12688487.v112.\n\nThis project contains the following extended data:\n\nTable 1- Health and social care interventions systematic review-aims, outcomes and conclusions.\n\nTable 2- Risk of bias of included studies.\n\nTable 3- Evidence map of health and social care interventions of included systematic reviews based on conditions and outcomes.\n\nTable 4- Evidence summary at systematic review level for efficacy for health and social care interventions by outcome.\n\nTable 5- Mixed methods evidence identified.\n\nAppendix 1- Example search strategy.\n\nAppendix 2- Example data extraction form.\n\nAppendix 3- PRISMA Flowchart.\n\nAppendix 4- Definitions of interventions.\n\nFigshare: PRISMA checklist (Appendix 5) for ‘Does health and social care provision for the community dwelling older population help to reduce unplanned secondary care, support timely discharge and improve patient well-being? 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde São José J, Barros R, Samitca S, et al.: Older persons' experiences and perspectives of receiving social care: a systematic review of the qualitative literature. Health Soc Care Community. 2016; 24(1): 1–11. PubMed Abstract \| Publisher Full Text\n\nPettersson C, Iwarsson S: Evidence-based interventions involving occupational therapists are needed in re-ablement for older community-living people: A systematic review. Br J Occup Ther. 2017; 80(5): 273–285. Publisher Full Text\n\nTessier A, Beaulieu MD, Mcginn CA, et al.: Effectiveness of Reablement: A Systematic Review. Efficacité de l'autonomisation: une revue systématique. Healthc Policy. 2016; 11(4): 49–59. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoniface G, Mason M, MacIntyre J, et al.: The Effectiveness of Local Authority Social Services’ Occupational Therapy for Older People in Great Britain: A Critical Literature Review. Br J Occup Ther. 2013; 76(12): 538–547. Publisher Full Text\n\nColl-Planas L, Nyqvist F, Puig T, et al.: Social capital interventions targeting older people and their impact on health: a systematic review. J Epidemiol Community Health. 2017; 71(7): 663–672. PubMed Abstract \| Publisher Full Text\n\nLeung P, Orrell M, Orgeta V: Social support group interventions in people with dementia and mild cognitive impairment: a systematic review of the literature. Int J Geriatr Psychiatry. 2015; 30(1): 1–9. PubMed Abstract \| Publisher Full Text\n\nCabrera E, Sutcliffe C, Verbeek H, et al.: Non-pharmacological interventions as a best practice strategy in people with dementia living in nursing homes. A systematic review. Geriatr Med. 2015; 6(2): 134–150. Publisher Full Text\n\nFolkerts AK, Roheger M, Franklin J, et al.: Cognitive interventions in patients with dementia living in long-term care facilities: Systematic review and meta-analysis. Arch Gerontol Geriatr. 2017; 73: 204–221. 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PubMed Abstract \| Publisher Full Text\n\nHagan R, Manktelow R, Taylor BJ, et al.: Reducing loneliness amongst older people: a systematic search and narrative review. Aging Ment Health. 2014; 18(6): 683–693. PubMed Abstract \| Publisher Full Text\n\nFranck L, Molyneux N, Parkinson L: Systematic review of interventions addressing social isolation and depression in aged care clients. Qual Life Res. 2016; 25(6): 1395–1407. PubMed Abstract \| Publisher Full Text\n\nShvedko A, Whittaker AC, Thompson JL, et al.: Physical activity interventions for treatment of social isolation, loneliness or low social support in older adults: a systematic review and meta-analysis of randomised controlled trials. Psychol Sport Exerc. 2018; 34: 128–137. Publisher Full Text\n\nChen YR, Schulz PJ: The Effect of Information Communication Technology Interventions on Reducing Social Isolation in the Elderly: A Systematic Review. J Med Internet Res. 2016; 18(1): e18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKachouie R, Sedighadeli S, Khosla R, et al.: Socially assistive robots in elderly care: a mixed-method systematic literature review. International Journal of Human-Computer Interaction. 2014; 30(5): 369–393. Publisher Full Text\n\nChipps J, Jarvis MA, Ramlall S: The effectiveness of e-Interventions on reducing social isolation in older persons: A systematic review of systematic reviews. J Telemed Telecare. 2017; 23(10): 817–827. PubMed Abstract \| Publisher Full Text\n\nMacdonald GM: Using Systematic Reviews to Improve Social Care. Bristol: The Policy Press and London: Social Care Institute for Excellence. 2003. Reference Source\n\nCoulter A, Collins A: Making shared decision-making a reality: no decision about me without me. The King's Fund, London. 2011. Reference Source\n\nPope C, van Royen P, Baker R: Qualitative methods in research on healthcare quality. Qual Saf Health Care. 2002; 11(2): 148–152. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNational Institute for Clinical Excellence: Transition Between Inpatient Hospital Settings and Community or Care Home Settings for adults with social care needs. NICE guidelines [NG27]. London. 2015. Reference Source\n\nElliott JH, Turner T, Clavisi O, et al.: Living systematic reviews: an emerging opportunity to narrow the evidence-practice gap. PLoS Med. 2014; 11(2): e1001603. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAgency for Healthcare Research and Quality: SRDR: Systematic Review Data Repository. 2013. Accessed 8 November 2013. Reference Source" }	[ { "id": "68566", "date": "27 Aug 2020", "name": "Clarice Tang", "expertise": [ "Reviewer Expertise Health service management", "chronic disease", "physical therapy" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 of the systematic reviews has been conducted with rigour and is well written. It does also provide important information about the lack of evidence in health and social care interventions which is in-line with the current level of evidence around this literature (Poupard et al. 2020).1\n\nMy only comment is to consider the potential lack of evidence in the lack of evidence in health and social care interventions may be due to other reasons apart from the lack of objective outcomes in social support which has not been highlighted in the discussion. Firstly, disease-specific management such as heart failure and COPD do have specific evidence-based clinical pathways which will likely result in a positive effect on medical management for these particular diseases as compared to older people with mixed conditions. Secondly, the type of care management differed greatly across the various studies. The length of interventions and time of implementation of care management may have resulted in a different outcome. Comments regarding if factors such as length of interventions and timing of interventions impacted on the final results will be helpful for the readers.\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": "95251", "date": "29 Sep 2021", "name": "Johannes Siegrist", "expertise": [ "Reviewer Expertise Medical sociology and social epidemiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-conducted meta-review on a complex topic with relevance to health care of older people. Methodologically, I do not see any need for change or additional analysis. In terms of content, the main finding indicates a continued poor emphasis on integrated analysis of health and social care (as well as a methodologically under-developed state of intervention research on effectiveness of social care). The conclusions of this review are justified, and they include some fruitful suggestions for further developments.\nIf I miss something relevant in this paper, it concerns the lack of discussing the impact of a country's health care system on innovations in integrating health and social care. For instance, what can the UK system learn from developments in other countries? Maybe authors are in a position to discuss this issue, given the accumulated evidence from their literature search.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-857
https://f1000research.com/articles/9-856/v1	31 Jul 20	{ "type": "Research Article", "title": "Global trends and current status in colistin resistance research: a bibliometric analysis (1973-2019)", "authors": [ "Abdourahamane Yacouba", "Ahmed Olowo-okere", "Ahmed Olowo-okere" ], "abstract": "Background: Colistin resistance is a major breach in our last line of defense and without urgent action, we are heading for a post-antibiotic era, in which common infections and minor injuries can once again kill. To the best of our knowledge, the use of the bibliometric analytical technique for examining colistin resistance-related research does not exist in the literature. Methods: Here, we analyze and present bibliometric indicators of the global literature in colistin resistance research. The Scopus database was searched for articles on colistin resistance. The articles retrieved were analyzed using the bibliometrix R-package. Results: A total of 1105 publications were retrieved. There was a noticeable increase in the number of publications on colistin resistance research in the past decade. Six journals made up the core zone in colistin research and produced 35.83% of the published articles. The analysis across time-intervals revealed several keywords that had increased or decreased in usage when comparing the interval between 1973-2009 and 2010-2019. Authors’ keywords “Acinetobacter baumanii”, and “Pseudomonas aeruginosa” were the most frequent encountered during the period of 1973-2009, while “mcr-1”, “Enterobacteriaceae”, “Escherichia coli”, and “Klebsiella pneumoniae” emerged in the past decade. Conclusions: There has been a significant growth in publications on colistin resistance in the past decade, suggesting an urgent need for action by different stakeholders to contain this threat of colistin resistance. Keyword analysis revealed temporal changes in the types of keywords used across time-intervals. These findings summarize a general vision on colistin resistance research and will serve as baseline data for future comparative purposes.", "keywords": [ "colistin resistance", "mcr-1", "keywords", "bibliometric analysis" ], "content": "Introduction\n\nAccording to the World Health Organization (WHO), antibiotic resistance is one of today's greatest threats to global health, food security, and development1. The emergence and unprecedented spread of carbapenemases have led to a resurgence in the use of last-line antibiotic colistin as salvage therapy2–5.\n\nColistin or polymyxin E is a polypeptide antibiotic developed in the 1950s. Originally, the antibiotic was isolated from the soil bacterium Paenibacillus polymyxa subsp6. Though withdrawn from clinical use in the 1980s owing to its significant toxicity, it has been re-introduced for the treatment of multidrug resistant (MDR) bacteria, particularly carbapenem-resistant Gram-negative bacterial infections4,5,7,8. It acts by interfering with bacterial cell membranes and/or the inhibition of bacterial respiration9,10.\n\nIn 2016, the first plasmid-mediated colistin resistance gene, mcr-1, was detected in Escherichia coli and Klebsiella pneumoniae11. Since then, several variants of mcr-1 and novel families of mcr genes have been identified in a variety of samples12–18. Colistin resistance is a major breach in our last line of defense and without urgent action, we are heading for a post-antibiotic era, in which common infections and minor injuries can once again kill.\n\nBibliometrics is the branch of library science that applies mathematical and statistical techniques to analyze articles, reviews, editorial letters, books, and other documents19. This is an important tool to assess new trends in scientific research. To the best of our knowledge, the use of the bibliometric analytical technique for examining colistin resistance-related research does not exist in the literature. Here, we analyze and present bibliometric indicators of the global literature in colistin resistance research.\n\n\nMethods\n\nArticles on colistin resistance published in the literature were retrieved from the Scopus database on November 15, 2019. Keywords used for data extraction were obtained from published review articles on colistin resistance20–22. The search query used for data extraction was: TITLE-ABS-KEY (\"colistin resistance\" OR \"polymyxin E resistance\").\n\nTwo authors independently performed the literature search using EndNote X9 (Bld 12062) produced by Clarivate Analytics. Papers published between January 1, 1973 and 31 December 2019 were included. After removal of duplicate results, titles and abstracts were reviewed for relevance to the research question. Any disagreement was resolved by consensus of the two authors. Experimental studies and studies that reported colistin sensitive bacteria were not eligible for inclusion. Searches were restricted to studies published in the English language.\n\nAccording to similar previously published bibliometric studies, selected documents were analyzed by different standard indicators including total number of publications, document types, countries/territories, authors, sources, total number of citations, h-index, average number of citations per publication, international collaborations and Bradford division analysis using the bibliometrix R-package23,24. Furthermore, graphs were plotted for visualization of co-citation networks and keyword co-occurrence of the text corpus extracted from the title and the abstract fields of the articles25. Co-citation has been defined as any two items (authors) that have been jointly cited by another item (author). To determine temporal changes in the type of keywords in colistin resistance research, analysis was carried out for two-time intervals of the study period: period 1, representing 1973 to 2009, and period 2, from 2010 to 2019. All data analysis was performed using the open-source bibliometrix R-package 26 on RStudio version 1.2.1335 (Boston, MA). The bibliometrix R-package provides a set of functions for quantitative research in bibliometric, including: (1) data loading and conversion to R dataframe (readFiles(), convert2df()), (2) data analysis (summary() and plot(), citations(), local citations(), Hindex(), lotka(), keywordGrowth(), keywordAssociation()), and (3) data visualization (biblioNetwork(), histNetwork())26.\n\n\nResults\n\nA total of 1578 non duplicate publications indexed in Scopus were retrieved on November 15, 2019. Of the 1578 studies, 1105 met the inclusion criteria and were used for the bibliometric analysis (Figure 1)27. The result of distribution by time indicates that the literature in this field was first published in 1973. A significant increase occurred in the number of publications from 35 in the period 1973-2009 to 1070 in the past decade (P value <0.001). Most of the publications were original research studies (n = 862; 78.01%), followed by editorial letters (n = 124; 11.22%), and reviews (n = 65; 5.88%).\n\nThe top 10 cited papers, their citation frequency and titles are listed in Table 1. The most-cited article (n = 1632) was written by Liu et al. (2016), followed by papers authored by Olaitan et al. (2014) (n = 412) and Moffatt et al. (2010) (n = 336). The top 10 preferred sources for publishing documents on colistin resistance are shown in Table 2. Bradford division analysis shows six sources in the core zone. These six sources are Antimicrobial Agents and Chemotherapy, Journal of Antimicrobial Chemotherapy, International Journal of Antimicrobial Agents, Frontiers in Microbiology, Journal of Global Antimicrobial Resistance, and The Lancet Infectious Diseases, which produced 35.83% of the published articles.\n\naBy the end of year 2018; TC, total citation.\n\nThe top 10 most productive territories included two Asian countries, seven European countries and one country in North America. China dominated the literature in colistin resistance research with 212 items (19.18%), followed distantly by the USA (n = 166; 15.02%) and France (n = 120; 10.85%). China also had the highest citation frequency (Figure 2). International collaboration analysis for active countries is shown in Figure 3.\n\nEach node in the figure symbolizes a different country’ collaboration with other countries. The node’s diameter corresponds to the strength of collaboration. Links represent international collaboration pathways between countries. Cluster of countries having similar cluster color most probably represents a closely related research group. Links represent the strength of collaboration.\n\nThe 1105 publications were written by 5141 authors working on this subject. Author analysis highlighted 0.215 documents per author, 4.65 authors per document, and 7.37 co-authors per document, with a collaboration index of 4.74. A co-citation network of authors showed four clusters: the blue cluster dominated by Liu 2016; the red cluster dominated by Olaitan 2014; the green cluster dominated by Falagas 2005 and the purple cluster dominated by Xavier 2016 (Figure 4).\n\nEach node in the network symbolizes a different author’ co-citation. The node’s diameter corresponds to the strength of co-citation. Links represent co-citation pathways between authors. Co-citation network of authors showed four clusters. Links diameter represent the strength of co-citation.\n\nTemporal changes in the type of keywords were observed between the period 1 (1973 to 2009) and period 2 (2010 to 2019) (Table 3).\n\n\nDiscussion\n\nThe aim of this study was to analyze the global scientific outputs of colistin resistance research and show the trends and current status in colistin resistance research. The publication trend showed a significant increase in the number of publications, particularly in the past decade, from 2010 to 2019, which corresponds with the peak clinical interest in its use as antibiotic of choice against MDR Gram-negative bacteria. This trend reflects the increasing attention paid by different stakeholders to concerns about colistin resistance worldwide and shows that research aiming to reduce the threat of colistin resistance is gaining attention in the scientific community.\n\nBradford division analysis showed a high concentration (35.83%) of publications in a few journals, namely Antimicrobial Agents and Chemotherapy, Journal of Antimicrobial Chemotherapy, International Journal of Antimicrobial Agents, Frontiers in Microbiology, Journal of Global Antimicrobial Resistance, and The Lancet Infectious Diseases. Four of these six sources are exclusively dedicated to antimicrobials, which is consistent with the global effort against antimicrobial resistance.\n\nChina was the leading country for publications related to colistin resistance, contributing 19.18% of all Scopus database articles published during the period of study. This may be due to the widespread use of colistin in animal feedstock and agriculture in China45. Moreover, the fact that China was the first country where a plasmid-mediated mechanism of colistin resistance, the mobilizable colistin resistance gene-1 (mcr-1), was reported, may be a contributing factor to China’s commitment11. The majority of publications on colistin resistance research were produced by high-income countries, with a negligible contribution from low- and middle-income countries, particularly Africa, no countries in which were on the list of the top ten most productive countries. Several previous bibliometric studies of the literature have also reported a similar distribution of research publications in different fields from high-income countries23,46,47. This poor research output from African countries could be due to the lack of funding and poor facilities.\n\nWith regards to keywords, the analysis across time-intervals revealed that several keywords have increased or decreased in usage when comparing the interval between 1973-2009 and 2010-2019. This shows a temporal change in the type of keywords. Keywords “mcr-1”, “Enterobacteriaceae”, “Escherichia coli”, and “Klebsiella pneumoniae”, which were absent from 1973 to 2009, have emerged in the past decade. This could be attributed to the fact that until the first report of mcr-1 gene, colistin resistance in Enterobacteriaceae was believed to be chromosomally mediated36. However, since 2016, the mcr-1 gene was reported in Enterobacteriaceae recovered from food, animals, and human specimens in China, and it has subsequently been reported worldwide11,48–53. The wide spread of the mcr-1 dramatically challenges the newly renewed interest in colistin for clinical use and opens a new research topic on colistin. Currently, eight new mcr genotypes (mcr-2 to mcr-9) have been reported since the discovery of mcr-114–18,33,54,55. Furthermore, colistin-resistant bacteria without prior colistin exposure, possibly due to cross-resistance between colistin and cationic antimicrobials such as LL-37 and lysozyme, have also been reported56,57.\n\nUnsurprisingly, the article “Emergence of plasmid-mediated colistin resistance mechanism mcr-1 in animals and human beings in China: a microbiological and molecular biological study,” published by the Lancet 11 was the most cited article.\n\nCountry collaboration in colistin resistance research remains relatively weak. This raises the fundamental question of global collaboration to slow the development and spread of colistin resistance58. Country collaboration may be the most practical solution to this transmissible plasmid-mediated resistance in the era of globalization, with increased migration, trade and travel. Indeed, horizontal transfer of mcr-mediated colistin resistance is a rapid phenomenon, and can not only occur at a rather high frequency but can also disseminate across different bacterial species. Following its first description, plasmid-mcr has now been reported across all seven continents59.\n\nTo the best of our knowledge, this study is the first to initiate baseline data on bibliometrics in colistin resistance research. The study was not limited by language and a large literature including original articles, reviews, editorial letters and meeting abstracts published over a long period was included. The Scopus database covers the vast majority of online research, particularly broader biomedical research, and it has been established that it is the most user-friendly and easiest tool to use for bibliometric analysis services60,61. Furthermore, comprehensive and relatively objective data analysis was performed, which clearly highlighted the past and current status of colistin resistance research and predicted the future research frontier.\n\nHowever, this global study is characterized by a number of limitations that are inherent in bibliometric analysis62. The main limitation of the present data is the reliance solely upon the Scopus database, which did not represent all the literature. Despite the fact that the Scopus database is considered an excellent source for bibliometric analysis, there are some journals that contain publications on colistin resistance but are not indexed in Scopus and therefore were not counted. Furthermore, we included papers where colistin resistance or polymyxin E resistance was used in the title or abstract or keywords; that inclusion of search items gives a much lower sensitivity to the search.\n\n\nConclusions\n\nThere has been a significant growth of publications on colistin resistance in the past decade, suggesting an urgent need for action by different stakeholders to contain this threat of colistin resistance. Unsurprisingly, an article on mcr-1 gene research ranked first among the top ten most cited articles on colistin resistance. Keyword analysis revealed temporal changes in the types of keywords used across time-intervals. These findings summarize a general vision on colistin resistance research and will serve as baseline data for future comparative purposes.\n\n\nData availability\n\nFigshare: Full list of included studies .bib. https://doi.org/10.6084/m9.figshare.12673151.v127.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgements\n\nThe authors thank Maxime Descartes Mbogning Fonkou (Aix Marseille University) for his collaboration in the preliminary data collection and valuable comments.\n\n\nReferences\n\nWHO: Global action plan on antimicrobial resistance. Resolution WHA68.7. Agenda item 15.1. 2015; [cited 20 May 2020]. Reference Source\n\nTrecarichi EM, Tumbarello M: Therapeutic options for carbapenem-resistant Enterobacteriaceae infections. Virulence. 2017; 8(4): 470–84. 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PubMed Abstract \| Publisher Full Text\n\nHuang X, Yu L, Chen X, et al.: High Prevalence of Colistin Resistance and mcr-1 Gene in Escherichia coli Isolated from Food Animals in China. Front Microbiol. 2017; 8: 562. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSweileh WM, Shraim NY, Al-Jabi SW, et al.: Bibliometric analysis of global scientific research on carbapenem resistance (1986–2015). Ann Clin Microbiol Antimicrob. 2016; 15(1): 56. Publisher Full Text\n\nSweileh WM, Al-Jabi SW, Zyoud SH, et al.: Global research output in antimicrobial resistance among uropathogens: A bibliometric analysis (2002–2016). J Glob Antimicrob Resist. 2018; 13: 104–14. PubMed Abstract \| Publisher Full Text\n\nGutiérrez C, Zenis J, Legarraga P, et al.: Genetic analysis of the first mcr-1 positive Escherichia coli isolate collected from an outpatient in Chile. Braz J Infect Dis. 2019; 23(3): 203–6. PubMed Abstract \| Publisher Full Text\n\nSulaiman AAA, Kassem II: First report on the detection of the plasmid-borne colistin resistance gene mcr-1 in multi-drug resistant E. coli isolated from domestic and sewer waters in Syrian refugee camps in Lebanon. Travel Med Infect Dis. 2019; 30: 117–20. PubMed Abstract \| Publisher Full Text\n\nRhouma M, Thériault W, Rabhi N, et al.: First identification of mcr-1/mcr-2 genes in the fecal microbiota of Canadian commercial pigs during the growing and finishing period. Vet Med (Auckl). 2019; 10: 65–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAgeevets V, Lazareva I, Mrugova T, et al.: IncX4 plasmids harbouring mcr-1 genes: Further dissemination. J Glob Antimicrob Resist. 2019; 18: 166–7. PubMed Abstract \| Publisher Full Text\n\nNewton-Foot M, Snyman Y, Maloba MRB, et al.: Plasmid-mediated mcr-1 colistin resistance in Escherichia coli and Klebsiella spp. clinical isolates from the Western Cape region of South Africa. Antimicrob Resist Infect Control. 2017; 6: 78. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTarabai H, Valcek A, Jamborova I, et al.: Plasmid-Mediated mcr-1 Colistin Resistance in Escherichia coli from a Black Kite in Russia. Antimicrob Agents Chemother. 2019; 63(9): e01266–19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang X, Wang Y, Zhou Y, et al.: Emergence of a novel mobile colistin resistance gene, mcr-8, in NDM-producing Klebsiella pneumoniae. Emerg Microbes Infect. 2018; 7(1): 122–122. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbuOun M, Stubberfield EJ, Duggett NA, et al.: mcr-1 and mcr-2 (mcr-6.1) variant genes identified in Moraxella species isolated from pigs in Great Britain from 2014 to 2015. J Antimicrob Chemother. 2017; 72(10): 2745–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNapier BA, Burd EM, Satola SW, et al.: Clinical Use of Colistin Induces Cross-Resistance to Host Antimicrobials in Acinetobacter baumannii. mBio. 2013; 4(3): e00021–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOlaitan AO, Morand S, Rolain JM: Emergence of colistin-resistant bacteria in humans without colistin usage: a new worry and cause for vigilance. Int J Antimicrob Agents. 2016; 47(1): 1–3. PubMed Abstract \| Publisher Full Text\n\nPiffaretti JC: Antibiotic resistance: the emergence of plasmid-mediated colistin resistance enhances the need of a proactive one-health approach. FEMS Microbiol Lett. 2016; 363(5): fnw034. PubMed Abstract \| Publisher Full Text\n\nNang SC, Li J, Velkov T: The rise and spread of mcr plasmid-mediated polymyxin resistance. Crit Rev Microbiol. 2019; 45(2): 131–61. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "70235", "date": "07 Sep 2020", "name": "David Lupande-Mwenebitu", "expertise": [ "Reviewer Expertise Clinical Microbiologist", "Antibiotics resistance", "Hygien and noscomial infections surveillance" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work is interesting, with this merit indeed of having used bibliometrics as a tool, however, several other databases must be consulted including Google Scholar, Web of Science and Pubmed to come out as large as possible. To work over a long period and to find only less than 2000 published papers seems insufficient to me. Currently, there are 10 types of mcr gene, mcr-10 was described last March1.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "74440", "date": "05 Jan 2021", "name": "Ismaeel Yunusa", "expertise": [ "Reviewer Expertise Research methodologist. Internal Medicine", "Oncology", "Geriatrics", "and Infectious diseases" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors used the bibliometric analytical technique to examine research related to colistin resistance. In the absence of any previous study on the current state of research for this problem, I find this study timely. However, I have the following comments for improvement:\n\nUnder the \"Strengths and limitations of research\" section, authors mentioned that \"the study was not limited by language\"; however, in the \"Methods\" Section, they mentioned that \"Searches were restricted to studies published in the English language.\". This means that a focus on English language articles actually limited the study. Authors may need to modify as appropriate.\n\nAuthors may need to mention how they handled articles with full-texts in another language, but abstract and other author characteristics are in English.\n\nAuthors may provide further context on a one-sentence paragraph in the discussion. Or consider incorporating the sentence into another paragraph, if appropriate.\n\nPresenting a graph, if possible, that illustrates the trend in publications over time might be helpful. The title itself reads global trends, so a graph showing temporal relationships 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? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-856
https://f1000research.com/articles/9-116/v1	14 Feb 20	{ "type": "Research Article", "title": "Transcranial direct current stimulation and attention skills in burnout patients: a randomized blinded sham-controlled pilot study", "authors": [ "Pia Van Noppen", "Kim van Dun", "Siel Depestele", "Stefanie Verstraelen", "Raf Meesen", "Mario Manto", "Pia Van Noppen", "Siel Depestele", "Stefanie Verstraelen", "Raf Meesen", "Mario Manto" ], "abstract": "Background: Burnout is characterized by deficiencies in attention and several components of the working memory, of which the lingering effects of impaired attention and executive functions are the most frustrating. We hypothesized that anodal transcranial direct current stimulation (atDCS) over the left dorsolateral prefrontal cortex (DLPFC) can improve the executive control of attention and possibly several other components of working memory in patients with burnout.\n\nMethods: This was a randomized double-blind sham-controlled pilot study with two groups. Patients with burnout received three weeks of daily sessions (15 sessions in total) of atDCS or sham stimulation in addition to three weekly sessions of standard behavioral therapy. The primary outcome measure was attention and the central executive of the working memory. Secondary, the effect of atDCS was measured on other components of working memory, on burnout and depression scores, and on quality of life (QoL).\n\nResults: We enrolled and randomly assigned 16 patients to a sham or real stimulation group, 15 (7 sham, 8 real) were included in the analysis. atDCS had a significant impact on attention. Post-hoc comparisons also revealed a trend towards more improvement after real tDCS for inhibition and shifting, updating and control, and encoding. Both groups improved on burnout and depression scores.\n\nConclusion: These data provide preliminary evidence for the value of atDCS over the left DLPFC in rehabilitating attention deficits, and possibly also central executive and encoding deficits, in burnout. However, the current study has some limitations, including the sample size and heterogeneous patient population. More elaborate studies are needed to elucidate the specific impact of atDCS over the left DLPFC on burnout.\n\nTrial registration: ISRCTN.com (ISRCTN94275121) 17/11/19", "keywords": [ "burnout", "direct current stimulation", "attention", "working memory", "prefrontal cortex" ], "content": "Abbreviations\n\natDCS anodal transcranial Direct Current Stimulation\n\nBDI Beck’s Depression Inventory\n\nBNT Boston Naming Test\n\nDSM-V 5th edition of the Diagnostic and Statistical Manual for Mental Disorders\n\nICD-10 10th edition of the International Statistical Classification of Diseases and Related Health Problems\n\nMBS Maslach Burnout Scale\n\nNMDA N-Methyl-D-Aspartate\n\nRBANS Repeatable Battery for the Assessment of Neuropsychological Status\n\nSD Standard Deviation\n\ntDCS transcranial Direct Current Stimulation\n\nTMT Trail Making Test\n\nWCST Wisconsin Card Sorting Test\n\nQoL McGill Quality of Life Questionnaire\n\n\nIntroduction\n\nThe percentage of employees experiencing burnout is dramatically increasing in Europe (Eurofound, 2018), which has a significant socio-economic impact. Burnout consists of three components: (1) exhaustion at the physical level (energy loss, fatigue, weakness, physical and psychosomatic complaints), the mental level (negative behavior towards oneself, work, or life in general), or the emotional level (feelings of being trapped in a situation, helplessness, or hopelessness); (2) depersonalization or alienation towards the actual work, towards patients or pupils, etc. (Demerouti et al., 2001; Schaufeli & Enzmann, 1998); (3) and reduced professional performance, which can be attributed to depersonalization and alienation (Demerouti et al., 2001). Since the beginning of 2011, burnout has been added to the 10th edition of the International Statistical Classification of Diseases and Related Health Problems (ICD-10: Z73.0: Word Health Organization, 2011), which describes burnout as a 'state of vital exhaustion'. The 5th edition of the Diagnostic and Statistical Manual for Mental Disorders (DSM-V: American Psychiatric Association, 2013), on the other hand, categorizes burnout under 'somatic symptoms and related disorders'.\n\nPatients with burnout are impaired in one or more of the four components of working memory, i.e. the central executive, the phonological loop, the visuospatial sketchpad and/or the episodic buffer (see Figure 1) (Baddeley, 2000; Deligkaris et al., 2014). The working memory, or the short-term memory, refers to a limited-capacity cognitive system that allows the temporary storage and manipulation of information from different modalities, provided by the sensory memory, that are necessary for complex tasks. (1) The phonological loop is responsible for encoding language in the long-term memory and for short-term retention of phonological information through repetition (Baddeley et al., 1998). (2) The visuospatial sketchpad temporarily stores visual and spatial information. (3) The episodic buffer temporarily stores and integrates information from the other components, and links information to time and space to make storage and invocation easier (Baddeley, 2000). These three components are controlled by the fourth component, i.e. (4) the central executive, which ensures that targeted actions can be taken by guiding attention towards relevant information in the sensory memory (Baddeley, 1996). The central executive operates by (1) inhibition, i.e. the suppression of dominant, automatic answers, and the resistance to interference caused by distractors; (2) shifting, which refers to the possibility to switch cognitively between various tasks, mental states, or operations; and (3) updating of the working memory (Miyake et al., 2000).\n\nThe working memory does not only monitor and direct attention, it is also responsible for the storage of information in the long-term memory (encoding) and recall of information from that same memory (retrieval) (Baddeley, 1996; Baddeley & Sala, 1996).\n\nBased on this model, deficits of executive functions and attention could be attributed to dysfunction of the central executive component (Baddeley, 1996). Accordingly, impairment of nonverbal memory deficits could be associated with the visuospatial sketchpad (Papagno, 2002), verbal memory deficits could be connected to the phonological loop (Vallar & Baddeley, 1984), and episodic (long-term) memory disruption could be attributed to dysfunction of the episodic buffer (Quinette et al., 2006). However, not all components of the working memory model are equally affected in burnout. A recent meta-analysis stated that burnout primarily affects attention, vigilance (i.e. sustained attention), and the central executive, more specifically memory updating and monitoring (Riedrich et al., 2017).\n\nTranscranial direct current stimulation (tDCS) is a non-invasive neurostimulation technique that modulates cortical excitability to enhance brain function by means of a low electrical current applied over the skull (Brunoni et al., 2012; Nitsche & Paulus, 2011). tDCS is increasingly used in the treatment of motor, cognitive, and affective symptoms in different patient populations, but also for treating a range of neuropsychiatric disorders, including Alzheimer’s disease and major depressive disorder (Brunoni et al., 2012; Flöel, 2014). The therapeutic potential of tDCS is gaining interest. In a double-blind sham-controlled trial consisting of three weeks (15 sessions) of active or sham anodal tDCS (atDCS) (2mA) over the left dorsolateral prefrontal cortex (DLPFC), Loo et al. confirmed the antidepressant efficacy of atDCS in patients with depression. In addition, mood, attention skills, and working memory also significantly improved after active tDCS treatment (Loo et al., 2012). Moreover, a recent study by Miler, Meron, Baldwin, and Garner showed that a single session of DLPFC stimulation can improve executive control of attention in healthy adults (Miler et al., 2018).\n\nStudies have shown that burnout patients are primarily impaired in attention and the central executive (Riedrich et al., 2017). We tested the hypothesis that atDCS over the left DLPFC could improve the general well-being of recovering burnout patients by boosting the recovery of the executive control of attention. Since this is the first study using tDCS in the rehabilitation of burnout patients, other components of the working memory were also measured to monitor the impact of burnout and the effect of atDCS on these components.\n\n\nMethods\n\nPatients were recruited between January 2015 and December 2017 via a treatment center in Belgium specialized in the diagnosis and treatment of burnout (DIADIS NV, Oud-Turnhout). The definition of (Brenninkmeijer et al., 2001) was used to identify burnout patients, and a score of > 4 on the Dutch version of the Maslach Burnout Scale (MBS: Maslach-Pines, 2005) was considered an inclusion criterium. Patients with 1) excessive drug or alcohol use, 2) epilepsy, 3) depression, 4) bipolar syndrome, 5) chronic fatigue syndrome or any other history of psychiatric or neurological disorders, 6) implanted neurostimulator or pace-maker, 7) drugs interacting directly with the NMDA receptors, or 8) pregnancy were excluded. When new patients were diagnosed with burnout in the treatment center, they were asked whether they wanted to participate in the study. Included patients were pseudo-randomly assigned to a real atDCS or sham tDCS group using a pre-defined allocation code file in excel (to make sure that both groups were of equal size). Initially, 20 participants were targeted (10 per tDCS group) as a pilot study. This number was primarily based on practical issues, such as the average number of burnout patients that were treated every year at the treatment center, and the time the treating psychologist could devote to the study. All assessments were performed by the sole psychologist of the treatment center (PVN).\n\nThis study was approved by the ethical committee CME of the Vrije Universiteit Brussels (VUB) (B.U.N. 143201422009). All patients signed an informed consent. The trial was retrospectively registered at ISRCTN.com on 17/11/19 (ISRCTN94275121), since clinical trial registration was not explicitly required by the advising ethical committee for trials with an experimental device at the start of the trial. All protocol and trial details are available from the registration page.\n\nAfter inclusion, baseline measures were taken to evaluate burnout, depression, quality of life, attention, and different components of the working memory. Burnout, depression, and overall quality of life were assessed by the MBS, the Beck’s Depression Inventory (BDI: Van der Does, 2002), and Question A of the Dutch version of the McGill Quality of Life Questionnaire (QoL: Cohen et al., 1997); translated by Kenniscentra Palliatieve Zorg) respectively.\n\nAttention was measured by the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS: Randolph, 1998) Attention Index, and vigilance by the s-score of the D2 test (variability in processing speed).\n\nThe central executive of the working memory was evaluated with the following tests. Inhibition and shifting were assessed with Card III of the Stroop Color-Word test (Golden, 1978), the Trail Making Test part B (TMT: Reitan, 1958) and the Wisconsin Card sorting test (WCST: Heaton et al., 1993). Processing speed, i.e., updating and control, was assessed by the TMT part A, Cards I and II of the Stroop Color-Word test, and the D2 test (Gz: total number of tokens scanned; F%: error percentage relative to Gz; Gz – F: number of correctly identified tokens) (Brickenkamp, 1962).\n\nAs regards to the other components of the working memory: the phonological loop was tested by the Language Index of the RBANS, the Boston naming test (BNT: Kaplan et al., 1983; Flemish version BNT: Mariën et al., 1998) and semantic fluency tasks (naming as many animals, vegetables, means of transportation and clothes as possible within one minute). To determine the percentile of semantic fluency, Dutch non-published age-, gender-, and education-related norms were used (These data were obtained by master students in Linguistics at the VUB of 200 healthy participants in Belgium of varying age, gender, education, and geographic location and are available as extended data (van Dun, 2020)). These data were used to calculate the z-scores that were then converted to percentiles. The visuospatial sketchpad was assessed using the Raven’s progressive matrices (Raven, 1965), and the Visuospatial Index of the RBANS. Encoding was evaluated with the Immediate Memory Index and retrieval with the Recent Memory Index of the RBANS.\n\nA categorized overview of the different tests is presented in Table 1.\n\nLegend: MBS = Maslach Burnout Scale; BDI = Beck’s Depression Inventory; QoL = McGill Quality of Life Questionnaire; RBANS = Repeatable Battery for the Assessment of Neuropsychological Status; Stroop = Stroop Color-Word test; TMT = TrailMaking Test; WCST = Wisconsin Card Sorting Test; BNT = Boston Naming Test; Raven = Raven’s progressive matrices; SS = Standard Score; Pct. = Percentile.\n\nAfter treatment, all tests were repeated to evaluate the impact of atDCS. Therapy always started on a Monday, and re-evaluation was completed the first Monday after the final atDCS session.\n\nThe primary outcome measure was attention. Secondary outcome measures were general measures (burnout, depression, and quality of life), and other components of the working memory (central executive, phonological loop, visuospatial sketchpad, encoding and retrieval).\n\nAll patients received the standard behavioral therapy consisting of one session a week (for 3 weeks) focusing on 1) psycho-education and relaxation, 2) reducing mental overload, 3) defining and working to personal goals, 4) relapse prevention. 1) In the first session, the stress mechanism was explained, together with the characteristics that belong to it. Breathing exercises were taught to the patient through heart rhythm coherence, using EmWave2 software to visually guide the patients. 2) To reduce the mental overload, ‘don’t worry’-techniques were explained. Patients were advised to write down their worries and not get distracted by them continuously. Via cognitive behavioral therapy, using the ABCDE model (Ellis et al., 1997), they were taught to translate negative into positive thoughts. 3) During therapy, the patient’s life goals in different domains (e.g. work, personal relations, education, parenthood, friends, physical well-being, …) were established together with the therapist. In dialogue, priorities were established and possible (mental) barriers were discussed. This discussion primarily focused on rebalancing the different domains in the patient’s life. 4) Lastly, the therapy focused on reintegration on the work floor. Bad habits were identified and strategies were discussed to prevent the patients from falling back into these habits.\n\nIn addition, patients received daily sessions of 2mA atDCS (TCT Research Limited, Hong Kong) over the left DLPFC (Fp3 on the international 10/20 EEG system) (electrode size: 5x5cm2) and the reference electrode (5x7cm2) over the lateral aspect of the contralateral orbit (F8), as described in (Loo et al., 2012). The carbon electrodes were covered in sponges soaked in saline solution (0.9% NaCl) to improve conductivity. In the real tDCS group, stimulation lasted for 20min with a gradual ramp up over 30s. This resulted in a maximal current density of 0.08mA/cm2 and a total charge of 0.096C/cm2 per session. Impedance was continuously monitored during stimulation to stay below 10kOhm and was automatically disrupted for safety when it went above 15kOhm. During sham stimulation, the current was ramped up over 30s to 2mA after which it was immediately ramped down to simulate the cutaneous sensation of tDCS in the sham group. No therapy was given during stimulation. This resulted in 15 sessions in total (3 weeks, 5x / week). One group received real tDCS, the other received sham tDCS. The tDCS device was programmed by the therapist, but the patients did not know which type of tDCS they received. All test results were coded to blind the researcher who performed the analyses and data was unblinded only after the analyses were done. The protocol and electrode placement are illustrated in Figure 2.\n\nA. Visualization of the protocol, and B. electrode placement of the tDCS protocol.\n\nAll therapy and tDCS sessions were performed at the treatment center DIADIS NV in Oud-Turnhout, where the patients were recruited, by the same psychologist and co-author Pia Van Noppen.\n\nMeans and standard deviations (SDs) were reported to give a general overview of the results. An independent samples t-test was used to compare mean age between groups.\n\nA full-factorial 2 (tDCS: sham, real) x 2 (time: pre, post) fixed effects linear mixed model with subject as a random effect was used to compare the results on all test measures between the sham and real tDCS groups before and after treatment. Normality and homoscedasticity of the residual data were checked via a normal quantile plot and residual plot, respectively. If model assumptions were violated, the outcome variable was transformed using the Box-Cox procedure (Box & Cox, 1964), as implemented in the MASS package in R version 7.3-51.4. Tukey HSD post-hoc pairwise comparisons were used to compare baseline scores between groups and to explore possible interaction effects. The level of significance was set at α = 0.05. All statistical analyses and figures were generated using the statistical software R version 3.6.0.\n\n\nResults\n\nIn total, 16 patients (11F, 5M) were recruited and received either real (n = 8) or sham (n = 8) treatment. Of these, 15 (10F, 5M) were included in the analysis. One participant (pp01, F, sham) was excluded after analysis because she was diagnosed with sensory processing sensitivity (SPS). Mean age of our final sample (n = 15) was 44.8y ± 5.8y, with no significant difference between the real (42.5y ± 5.5y) and sham group (47.4y ± 5.3y) (t(12.86) = 1.76, p = 0.103). No participants reported serious adverse events. Only one complained about dizziness at the end of the stimulation.\n\nAn overview of the demographic characteristics and the initial scores on the MBS, the Dutch version of the BDI, and question A of the Dutch version of the QoL are given in Table 2 (see underlying data (van Dun, 2020)). A flow chart is provided in Figure 3.\n\nLegend: F = Female; M = Male; MBS = Maslach Burnout Scale; BDI = Beck’s Depression Inventory; tDCS = transcranial Direct Current Stimulation; QoL = McGill Quality of Life Questionnaire; SD = Standard Deviation.\n\nThe linear mixed model revealed a significant effect of Time for the MBS (F(1, 13) = 15.10, p = 0.002), but no effect of tDCS (F(1, 13) = 0.00, p = 0.971) or an interaction (F(1, 13) = 0.73, p = 0.408) (see Figure 4A). Tukey HSD post-hoc multiple comparisons indicated that only the real tDCS group improved significantly on the MBS (real: t(13) = 3.886, p = 0.009; sham: t(13) = 2.465, p = 0.113).\n\nMean pre- and postscores on A. the Maslach Burnout Scale (MBS), B. Beck’s Depression Inventory (BDI), and C. McGill Quality of Life (QoL) questionnaire for the sham (dotted, triangles) and real (dashed, circles) group with 95% confidence intervals. Continuous lines indicate main effects, dashed and dotted lines indicate a significant difference between the pre- and postscores of the separate group (real: dashed; sham: dotted) as found by the post-hoc analyses. NS = non-significant; * = p≤0.05; ** = p≤0.01.\n\nFor the BDI, only an effect of Time was found ((F(1, 13) = 7.93, p = 0.015) (see Figure 4B). Post-hoc analyses revealed that only the sham group improved significantly after the intervention (t(13) = 3.58, p = 0.016) and the real group demonstrated a tendency towards improvement (t(13) = 2.82, p = 0.062).\n\nThe linear mixed model revealed no significant effects or interaction for the McGill Quality of Life (QoL) questionnaire. However, post-hoc analysis did reveal a significant improvement for the real group (t(13) = -3.21, p = 0.031) (see Figure 4C).\n\nNo significant differences were found at baseline for these three measures (MBS: t(13) = 0.04, p = 1.000; BDI: t(13) = -0.73, p = 0.883; QoL: t(13) = 0.00, p = 1.000). All means, SDs, and p-values of the post-hoc analyses are listed in Table 3. The results of the linear mixed model can be found in Table 7.\n\n* : p≤0.05\n\n*: p≤0.01\n\nLegend: tDCS = transcranial Direct Current Stimulation; MBS = Maslach Burnout Scale, BDI = Beck’s Depression Inventory; QoL = Quality of Life questionnaire.\n\nMeans, standard deviations, and p-values of the post-hoc analyses are shown in Table 4. The linear mixed model revealed a significant interaction between Time and tDCS for the RBANS Attention Index (F(1,13) = 14.80, p = 0.048), where the real group improved significantly more than the sham group (real: t(13) = -3.85, p = 0.010; sham: t(13) = -0.61, p = 0.929) (see Figure 5A). No significant difference was detected in the baseline scores (t(13) = 0.36, p = 0.984).\n\n : p≤0.05\n\n*: p≤0.01\n\nLegend: tDCS = transcranial Direct Current Stimulation; RBANS = Repeatable Battery for the Assessment of Neuropsychological Status; # = assumptions of the linear mixed model are violated.\n\nContinuous lines with X indicate interaction effects, dashed and dotted lines indicate a significant difference between the pre- and postscores of the separate group (real: dashed; sham: dotted) as found by the post-hoc analyses. X = interaction effect; NS = non-significant; = p≤0.05; ** = p≤0.01.\n\nA significant interaction effect was also found for vigilance (F(1,13) = 12.15, p = 0.004), as measured by the s-score of the D2 test, with the sham group improving significantly. However, the assumptions of homoscedasticity and normality of the residuals of the model were doubtful, but did not improve using the Box-Cox transformation, which makes it difficult to interpret the results. In addition, the real and sham tDCS group tended to differ significantly at baseline (t(13) = 2.82, p = 0.061), with the sham group performing worse than the real tDCS group.\n\nAll means, standard deviations, and p-values of the post-hoc analyses are shown in Table 5.\n\n* : p≤0.05\n\n** : p≤0.01\n\nLegend: tDCS = transcranial Direct Current Stimulation; TMT = Trail Making Test; WCST = Wisconsin Card Sorting Test; # = assumptions of the linear mixed model are violated.\n\nThe linear mixed model demonstrated a significant effect of Time for inhibition and shifting on the Stroop Color-Word test (card III) (F(1,13) = 5.96, p = 0.030) (Figure 6A) and the WCST (F(1,13) = 9.20, p = 0.010) (Figure 6B). Post-hoc comparisons only revealed a significant improvement in the real tDCS group on the WCST (real: t(13) = -3.03, p = 0.042; sham: t(13) = -0.54, p = 0.948). For the Stroop (card III) no significant improvements were found for either group post-hoc (real: t(13) = -2.44, p = 0.118; sham: t(13) = -0.53, p = 0.951). For the TMT B, the assumption of homoscedasticity of the residuals was violated and did not improve using the Box-Cox transformation, making interpretation of the model difficult. No significant effects or interaction were found with the non-transformed data.\n\nMean pre- and postscores for inhibition and shifting on A. the Stroop Color-Word test Card III, and B. the Wisconsin Card Sorting Test (WCST), for the sham (dotted, triangles) and real (dashed, circles) group with 95% confidence intervals. Continuous lines indicate main effects, dashed and dotted lines indicate a significant difference between the pre- and postscores of the separate group (real: dashed; sham: dotted) as found by the post-hoc analyses. NS = non-significant; * = p≤0.05; ** = p≤0.01.\n\nNo significant differences were found in the baseline measures (Stroop card III: t(13) = -1.78, p = 0.327; WCST: t(13) = -1.79, p = 0.323; TMT B: t(13) = 0.54, p = 0.947).\n\nFor updating and control, a significant effect of Time was found for the TMT A (F(1,13) = 7.69, p = 0.016) (Figure 7A), the Stroop Color-Word test (card I) (F(1,13) = 6.30, p = 0.026) (Figure 7B) and the D2 (Gz: F(1,13) = 7.38, p = 0.018; and Gz – F: F(1,13) = 8.10, p = 0.014) (Figure 7C and 7D). The post-hoc tests only revealed trends towards improvement in the real group for the TMT A (real: t(13) = -2.77, p = 0.067; sham: t(13) = -2.00, p = 0.238), the Stroop Color-Word test (card I) (real: t(13) = -2.51, p = 0.105; sham: t(13) = -0.24, p = 0.995), D2 Gz (real: t(13) = -2.72, p = 0.074; sham: t(13) = -1.02, p = 0.741), and D2 Gz – F (real: t(13) = -2.85, p = 0.059; sham: t(13) = -0.89, p = 0.812). No significant effects or interaction was found for the Stroop Color-Word test (card II) or F% of the D2 test. However, the assumption of homoscedasticity of the residuals was violated in the Stroop Color-Word test (card II), which might have resulted in unreliable p-values.\n\nMean pre- and postscores for updating and control on the A. Trail Making Test (TMT) part A, B. Stroop-Color Word test Card I, C. D2 Gz score, and D. D2 Gz – F score for the sham (dotted, triangles) and real (dashed, circles) group with 95% confidence intervals. Continuous lines indicate main effects, dashed and dotted lines indicate a significant difference between the pre- and postscores of the separate group (real: dashed; sham: dotted) as found by the post-hoc analyses. NS = non-significant; * = p≤0.05; ** = p≤0.01.\n\nNo significant differences were found in the baseline measures (TMT A: t(13) = 0.62, p = 0.925; Stroop card I: t(13) = -1.11, p = 0.689; Stroop card II: ; D2 Gz: t(13) = -0.97, p = 0.771; D2 Gz – F: t(13) = -1.27, p = 0.596; D2 F%: t(13) = -0.52, p = 0.952).\n\nAll mean scores, standard deviations, and p-values of the post-hoc analyses are listed in Table 6.\n\n: p≤0.05\n\n: p≤0.01\n\nLegend: tDCS = transcranial Direct Current Stimulation; RBANS = Repeatable Battery for the Assessment of Neuropsychological Status; BNT = Boston Naming Test; # = assumptions of the linear mixed model are violated.\n\nFor the phonological loop, the linear mixed model revealed a main effect of Time for the BNT (F(1,13) = 12.92, p = 0.003) (Figure 8A) and the Language index of the RBANS (F(1,13) = 4.76, p = 0.048) (Figure 8B). Tukey HSD post-hoc comparisons revealed that only the real tDCS group improved significantly on the BNT (t(13) = -3.60, p = 0.015), a trend towards improvement was observed in the sham group (t(13) = -2.83, p = 0.060). No significant improvements were found for the groups separately for the RBANS Language Index (real: t(13) = -2.18, p = 0.180; sham: t(13) = 0.78, p = 0.863). No significant effects or interactions were seen for semantic fluency. Baseline scores did not differ significantly for these measures (BNT: t(13) = 0.23, p = 0.995; RBANS Language Index: t(13) = 1.72, p = 0.352; Semantic fluency: t(13) = 0.63, p = 0.921).\n\nMean pre- and postscores for the phonological loop on the A. Boston Naming Test (BNT), and B. RBANS Language index for the sham (dotted, triangles) and real (dashed, circles) group with 95% confidence intervals. Continuous lines indicate main effects, dashed and dotted lines indicate a significant difference between the pre- and postscores of the separate group (real: dashed; sham: dotted) as found by the post-hoc analyses. NS = non-significant; = p≤0.05; ** = p≤0.01.\n\nNo significant effects or interaction were observed for the visuospatial sketchpad (RBANS Visuospatial index and Raven). However, the assumptions for the linear mixed model of the Raven were violated, which could have affected the p-values. The Box-Cox transformation did not improve the data. Baseline scores did not differ significantly (RBANS Visuospatial Index: t(13) = 1.72, p = 0.452; Raven: t(13) = -0.16, p = 0.999).\n\nA main effect of Time was observed for encoding, evaluated by the Immediate Memory index of the RBANS (F(1,13) = 11.93, p = 0.004) (Figure 9A). Post hoc analysis showed that this was mainly driven by a significant improvement of the real tDCS group (real: t(13) = -3.45, p = 0.020; sham: t(13) = -1.42, p = 0.508). Retrieval (RBANS Recent Memory index) also showed a significant effect of Time (F(1,13) = 8.58, p = 0.012) (Figure 9B). For retrieval, both groups trended towards significance (real: t(13) = -2.93, p = 0.051; sham: t(13) = -2.58, p = 0.094). No differences in baseline scores were observed for Immediate or Recent Memory (RBANS Immediate Memory Index: t(13) = 0.34, p = 0.986; RBANS Recent Memory Index: t(13) = -0.56, p = 0.943).\n\nMean pre- and postscores for encoding on the A. Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) Immediate Memory, and retrieval on the B. RBANS Recent Memory for the sham (dotted, triangles) and real (dashed, circles) group with 95% confidence intervals. Continuous lines indicate main effects, dashed and dotted lines indicate a significant difference between the pre- and postscores of the separate group (real: dashed; sham: dotted) as found by the post-hoc analyses. NS = non-significant; * = p≤0.05; ** = p≤0.01.\n\n: p≤0.05\n\n*: p≤0.01\n\nLegend: tDCS = transcranial Direct Current Stimulation; MBS = Maslach Burnout Scale; BDI = Beck’s Depression Inventory; RBANS = Repeatable Battery for the Assessment of Neuropsychological Status; TMT = Trail Making Test; WCST = Wisconsin Card Sorting Test; BNT = Boston Naming Test; # = assumptions of the linear mixed model are violated.\n\n\nDiscussion\n\nThis randomized blinded sham-controlled study investigated the impact of daily atDCS sessions (2mA, 20min) over the left DLPFC (Fp3) with the reference over the contralateral orbit (F8) on attention and the central executive, as well as other components of the working memory in patients with burnout. This electrode montage has been shown to be effective in patients with depression, showing not only an antidepressant effect but also a positive effect on mood, attention skills, and working memory (Loo et al., 2012). We included 15 patients (7 sham, 8 tDCS) in a 3-week protocol and investigated their cognitive and attention skills, as well as their burnout severity, depression, and overall quality of life before and after treatment. Both groups improved on all these measures, but the improvement of burnout and overall quality of life was only significant after real tDCS. Surprisingly, however, only the sham group significantly improved on the depression scale. This might be due to the fact that depression scores were moderate, while in the study of Loo et al. only patients with a DSM IV major depression episode were included (Loo et al., 2012). Moreover, in the study of Loo et al. depression was rated by an experienced psychiatrist/psychologist using the Montgomery Asberg Depression Rating Scale (MADRS; Montgomery & Asberg, 1979), while in our study a self-assessment scale (BDI) was used (Loo et al., 2012).\n\nFor the main variable of interest (Attention index of the RBANS), a significant interaction between tDCS and Time was found, showing that real anodal tDCS over the left DLPFC can have an added value to conventional therapy in the rehabilitation of attention in burnout patients.\n\nIt is known that burnout primarily impairs functions of the central executive, whereas brain areas that regulate other components of the working memory are affected to a lesser degree. The central executive -mainly located in the prefrontal brain regions- could be the component that is most vulnerable to chronic stress because its higher order attention control functions are more demanding and complex than those performed by the other subcomponents (Deligkaris et al., 2014). Several studies investigating the impact of burnout on cognitive functions have confirmed that the most pronounced differences between patients and controls were seen on tests that are highly dependent upon the executive functions, e.g. prospective memory, processing speed, complex working memory, sustained attention, and letter fluency (Eskildsen et al., 2015; Jonsdottir et al., 2013; Öhman et al., 2007; Orena et al., 2013)(35–38)(Eskildsen et al., 2015; Jonsdottir et al., 2013; Öhman et al., 2007; Orena et al., 2013). This study showed that three weeks of therapy, combined with real or sham stimulation, significantly improved several components of the central executive. However, analyses revealed that improvement was driven by a significant improvement after real tDCS for inhibition and shifting (WCST), and was also primarily seen after real tDCS for updating and control (TMT A, D2 Gz, D2 Gz – F). These results are in line with the study of Miler et al. who found that atDCS over the left DLPFC significantly improves the executive control of attention (Miler et al., 2018). As in the study of Miler et al., no effect was seen on the percentage of mistakes, but the processing speed did improve more on the D2 test of attention after real tDCS than after sham tDCS (Miler et al., 2018).\n\natDCS also seemed to have a positive impact on other components of the working memory. The phonological loop might also be positively influenced by real tDCS as shown by a significant improvement of the real tDCS group on the BNT, although the sham group also trended towards a significant improvement. No effect was seen on the visuospatial sketchpad, but encoding clearly improved more after real atDCS than after sham tDCS (RBANS Immediate Memory Index).\n\nThese data provide preliminary evidence for the value of tDCS over the left DLPFC in rehabilitating attention deficits, and possibly also central executive and encoding deficits, in burnout patients.\n\n\nLimitations and conclusion\n\nOur study has several important limitations. First, our group of patients was relatively small. This is an important limitation given the positive trends of the effect of real tDCS on several outcome measures. Studies with more power will have to show whether these trends failed to reach significance due to a lack of power. In addition, some variables of interest (D2 s-score, TMT B, Stroop Card II, Raven) could not be interpreted correctly with the linear mixed model analysis because of a violation of assumptions. More data points could help to resolve this issue.\n\nSecond, patients were randomized over both groups, which led to an overrepresentation of men in the sham group. At the moment, it is not clear whether gender can have a significant impact on the effect of tDCS (Antal et al., 2017), or whether there are gender-related differences in the symptoms of burnout (Purvanova & Muros, 2010), but this imbalance of gender between both groups might have affected the results.\n\nThird, our group of patients was very heterogeneous. For example, the moment of participation in the study was variable during the burnout process. Some participants were still at work, others were not yet able to start working, others were already re-integrated in their jobs. Due to the sample size, it was not possible to investigate the effects of different factors, such as living circumstances, age, gender, education, etc. on the progress of burnout. Future studies should focus on these parameters to elucidate the influence of these factors on burnout recovery and on tDCS outcome.\n\nDespite these shortcomings, these data provide preliminary evidence for the value of atDCS over the left DLPFC in rehabilitating attention deficits in burnout. tDCS might prove to be a useful, affordable, and easy-to-use addition to conventional therapy to speed up reintegration of burnout patients.\n\n\nConsent\n\nWritten informed consent for publication of the patients’ details was obtained from the patients.\n\n\nData availability\n\nHarvard Dataverse: Transcranial direct current stimulation and attention skills in burnout patients: a randomized blinded sham-controlled pilot study. https://doi.org/10.7910/DVN/4VG2XS (van Dun, 2020)\n\nThis project contains the following underlying data:\n\n- Data_burnout.txt (Data used for statistical analyses)\n\n- Raw Data Burnout.tab (Raw data for the Burnout study (Raw Scores (RS) and Standard Scores (SS)))\n\nHarvard Dataverse: Transcranial direct current stimulation and attention skills in burnout patients: a randomized blinded sham-controlled pilot study. https://doi.org/10.7910/DVN/4VG2XS (van Dun, 2020)\n\nThis project contains the following extended data:\n\n- Verbal_fluency.pdf (Means and standard deviations per age, gender, and educational level of 200 Dutch-speaking participants for the verbal (semantic) fluency task)\n\nCONSORT checklist and flow chart for “Transcranial direct current stimulation and attention skills in burnout patients: a randomized blinded sham-controlled pilot study”. https://doi.org/10.7910/DVN/4VG2XS (van Dun, 2020)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nWe also want to dedicate this work to Prof. dr. 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Harvard Dataverse. 2020. http://www.doi.org/10.7910/DVN/4VG2XS\n\nWord Health Organization: The ICD-10 classification of mental and behavioural disorders: clinical descriptions and diagnostic guidelines. (10th Edition). Geneva: World Health Organization, 2011. Reference Source" }	[ { "id": "63825", "date": "03 Jun 2020", "name": "Choi Deblieck", "expertise": [ "Reviewer Expertise non-invasive neuromodulation (TMS/tDCS)", "fMRI" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n\"Transcranial direct current stimulation and attention skills in burnout patients: a randomized blinded sham-controlled pilot study\" is addressing an important research and clinical question regarding executive memory, attention, and clinical symptoms in burnout. Data from 15 patients, randomized to 3 weeks of active anodal tDCS over left DLPFC or sham combined with standard behavioral therapy revealed that anodal tDCS significantly improved attention in patients with burnout.\n\nMajor Comments:\n\nMajor problems with neuromodulation studies are the small sample sizes and heterogeneous patient populations. The authors already mentioned those pitfalls as their biggest shortcomings. Nonetheless, it remains a major point of criticism for me. In order to get significance in TMS (and tDCS studies), Sack et al., (2009)1 demonstrated that power analyses showed 5 participants are sufficient to reveal a significant behavioral effect on cognition in TMS studies using fMRI-guided neuronavigation. However, the number of necessary participants increases to n = 9 in studies using MRI-guided neuronavigation, to n = 13 when using group Talairach coordinates, and to n = 47 when using EEG positions, like F3 or AF3.\n\nAs for the limitation of using heterogeneous patient groups, the demographic characteristics revealed are limited to age, gender, and burnout severity expressed by MBS. Reporting other relevant characteristics, such as duration, employment, ON/OFF medication, etiology, level of education, hospitalization, etc,… would give us a better understanding of how representative the groups were. Also, some of these variables could be explored for significance or even predictivity in future studies.\nAlso, since a trend towards inhibition and shifting was reported, and the right inferior frontal gyrus has been associated with inhibition, the choice of placing the cathode on F8 was not optimal. rIFG is only about 2 to 2.5 cm posterior to F8. Since the reference sponge used had a size of 5x7, rIFG could have been affected by the current.\n\nOnly continuous outcome measures were reported. Since it better reflects better clinical practice, I would add a categorical outcome measure, i.e., percentage of responders/remitters.\n\nA TCT device was used. The strap it comes with is made of a neoprene strap which could absorb the saline solution potentially increasing the surface area of the target area. Also, is this device EC approved?\n\nIt was unclear whether all the patients in both conditions had been undergoing psychotherapy prior to the add-on tDCS therapy. If psychotherapy was new to all, it is a major confound. It could also be the reason why both groups improved burnout and depression-wise.\n\nIn the tDCS protocol section, Fp3 is mentioned as the target site, “left DLPFC”. Since the 10-20 system of electrode placement is used, defining the target site as AF3 is probably better than Fp3. Also, the vast majority of TMS/tDCS studies targeting left DLPFC to stimulate F3. Why was Fp3/AF3 selected? DLPFC is indeed a large area. But an elaboration on the reason why a more anterior part of DLPFC was chosen should be explained.\n\nMinor Comments:\n\nIn the background section of the abstract/methods, only one objective (attention and executive control) is mainly elaborated upon. I would also add the clinical objective, i.e., burnout, depression amelioration. Only QoL was briefly mention.\n\nThe tDCS section of the introduction is not well structured. It’s better to subdivide the use of tDCS into neurological and psychiatric conditions. Now a distinction is made between “motor, cognitive, affective disorders” and “Alzheimer’s and MDD”.\n\nTwo papers on depression are mentioned: one submitting patients to 1 session and the other to 15 sessions. I would talk about the importance of the number of sessions as a potential variable that could increase tDCS effect (Brunoni)2 to explain why you submitted the patients to 15 sessions. Also, there are maybe one or two articles on stress in professionals, indirectly referring to burnout with no tDCS effect on burnout. One of the contributions of this article is that it may be the first looking at burnout. I would highlight this.\n\nI would also go more into details of other potential brain regions that could have been targeted, especially for those tasks that did not show a tDCS effect. Only DLPFC is mentioned. E.g. as mentioned above, inhibition has not been linked to DLPFC. Or why you included those tasks while targeting DLPFC. It could be the reason why no significance was observed in inhibition.\n\nA paragraph on neurotransmitters could also be added. Specifically, alterations in dopamine and noradrenaline levels in DLPFC have been associated with impaired working memory performance. TMS and tDCS over DLPFC have also been shown to release dopamine in various brain regions, including the right ventral striatum. Further, tDCS to DLPFC has shown to improve participants’ memory accuracy, an effect that has been correlated significantly with dopamine release.\n\nI would also mention briefly why it took 3 years to recruit 15 patients. It could be helpful for future studies recruiting patients with burnout.\n\nWere the patients asked at the end of the treatment whether they knew they had received sham or real? (chi-squared test to test the integrity of masking).\n\nWere patients asked to evaluate their level of discomfort? The level of discomfort/pain has been associated with a TMS/tDCS effect.\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": "5758", "date": "31 Jul 2020", "name": "Kim van Dun", "role": "Author Response", "response": "\"Transcranial direct current stimulation and attention skills in burnout patients: a randomized blinded sham-controlled pilot study\" is addressing an important research and clinical question regarding executive memory, attention, and clinical symptoms in burnout. Data from 15 patients, randomized to 3 weeks of active anodal tDCS over left DLPFC or sham combined with standard behavioral therapy revealed that anodal tDCS significantly improved attention in patients with burnout. Major Comments: Major problems with neuromodulation studies are the small sample sizes and heterogeneous patient populations. The authors already mentioned those pitfalls as their biggest shortcomings. Nonetheless, it remains a major point of criticism for me. In order to get significance in TMS (and tDCS studies), Sack et al., (2009)1 demonstrated that power analyses showed 5 participants are sufficient to reveal a significant behavioral effect on cognition in TMS studies using fMRI-guided neuronavigation. However, the number of necessary participants increases to n = 9 in studies using MRI-guided neuronavigation, to n = 13 when using group Talairach coordinates, and to n = 47 when using EEG positions, like F3 or AF3. Response: This is indeed a major shortcoming of our study but unfortunately, as a private practice, we do not have the facilities to set up a big study. Nonetheless, we do think our results are noteworthy and we therefore referred to our study as a “pilot study” hoping to instigate more interest in burnout in the field of non-invasive stimulation. In addition, since tDCS is not as focal as TMS, we do not think the use of EEG positions instead of neuronavigation will impact the power of the study as much as for TMS. As for the limitation of using heterogeneous patient groups, the demographic characteristics revealed are limited to age, gender, and burnout severity expressed by MBS. Reporting other relevant characteristics, such as duration, employment, ON/OFF medication, etiology, level of education, hospitalization, etc,… would give us a better understanding of how representative the groups were. Also, some of these variables could be explored for significance or even predictivity in future studies.Response: We did collect other demographic characteristics and have added these to the article to help future studies unravel the significance of these characteristics. Table 2B: Demographic characteristics and working-related information shows Employment (parttime or fulltime), Education level (12y of >12y), #working hours at intake, Smokers (yes or no), Living together / Children (yes or no), and Medication. Since three patients were on antidepressants (SSRI, SNRI, or SARI), of whom one (SSRI) received real stimulation, it has also been added to the limitations section that this might have impacted the outcome of this patient since it has been shown that chronic use of SSRIs can enhance the effect of tDCS.“In addition, three of the patients were taking antidepressant medication during the study, of whom one received real stimulation. It has been shown that this type of medication (selective serotonin reuptake inhibitors or SSRIs) might enhance the LTP-like plasticity induced by anodal tDCS (Kuo et al., 2016).”Also, since a trend towards inhibition and shifting was reported, and the right inferior frontal gyrus has been associated with inhibition, the choice of placing the cathode on F8 was not optimal. rIFG is only about 2 to 2.5 cm posterior to F8. Since the reference sponge used had a size of 5x7, rIFG could have been affected by the current. Response: This is a very good point that we have now addressed in the discussion. We based the placement of the electrodes on the study of Loo et al. (2012), who initially wanted to study the anti-depressant effect of tDCS but found an improvement of attention and working memory. Therefore, the electrode placement might not be optimal for working memory and attention. We have added this to the limitations section of the discussion.“Fourth, the placement of the electrodes might not have been optimal to target attention deficits. Our study was based on the outcome of Loo et al. (2012) who aimed to investigate the anti-depressant effect in patients with depression, but found an improvement of attention and working memory instead (Loo et al., 2012). By copying this electrode placement, we hoped to replicate these results in patients with burnout. However, by placing the cathode on F8, we might have unwantedly inhibited the right inferior frontal gyrus, which has been linked to inhibition and attentional control (Hampshire et al., 2010). Although cathodal tDCS over the right inferior frontal gyrus did not appear to have a significant effect on response stopping or reaction times in a stop-signal task (Stramaccia et al., 2015), another choice for the cathodal reference electrode might be warranted. In addition, AF3 targets primarily the more frontal site of the left DLPFC, while a more common placement to target DLPFC in attention studies is F3 (Coffman et al., 2014).”Only continuous outcome measures were reported. Since it better reflects better clinical practice, I would add a categorical outcome measure, i.e., percentage of responders/remitters. Response: Although we agree that, clinically, this would have added value, we do not really have a theoretical base for categorizing our participants into responders/remitters based on the outcome measures we used. Therefor we prefer to use the continuous scores for these outcome measures. A TCT device was used. The strap it comes with is made of a neoprene strap which could absorb the saline solution potentially increasing the surface area of the target area. Also, is this device EC approved? Response: The TCT device is not EC approved but it was thoroughly tested in the lab of prof. dr. Mario Manto and approved by our ethical committee. The neoprene strap indeed can absorb the saline solution but during stimulation it was checked whether this absorption spread beyond the electrode surface. In addition, both electrodes were attached to the scalp using different straps to avoid bridges between the electrodes through the straps. This has now been added to the methodology: “These were placed over the scalp using neoprene straps. Since these can absorb the saline solution, two different straps were used for both electrodes to avoid creating bridges and throughout the sessions the absorption was monitored so that it would not spread beyond the surface of the electrodes.” It was unclear whether all the patients in both conditions had been undergoing psychotherapy prior to the add-on tDCS therapy. If psychotherapy was new to all, it is a major confound. It could also be the reason why both groups improved burnout and depression-wise. Response: Psychotherapy was new to all, therefore we did expect a significant improvement, burnout- and depression-wise, in both groups. We have added this also to the text. However, our main focus for the effect of tDCS was on the attention component, since personal experience learned that attention deficits are the most resilient symptom of burnout. Methodology: “None of the patients had received psychotherapy before inclusion in this study.”Discussion: “Both groups improved on all these measures, which can be expected due to the behavioral therapy both groups received, but the improvement of burnout and overall quality of life was only significant after real tDCS.” In the tDCS protocol section, Fp3 is mentioned as the target site, “left DLPFC”. Since the 10-20 system of electrode placement is used, defining the target site as AF3 is probably better than Fp3. Also, the vast majority of TMS/tDCS studies targeting left DLPFC to stimulate F3. Why was Fp3/AF3 selected? DLPFC is indeed a large area. But an elaboration on the reason why a more anterior part of DLPFC was chosen should be explained. Response: As mentioned above, we based the placement of the electrodes on the study of Loo et al. (2012), who initially wanted to study the anti-depressant effect of tDCS. Therefore, this might indeed not be the ideal stimulation site for our purposes, but it has been proven to be effective in Loo et al. (2012). This has been added in the limitations section. We also changed Fp3 to AF3 as suggested. “In addition, AF3 targets primarily the more frontal site of the left DLPFC, while a more common placement to target DLPFC in attention studies is F3 (Coffman et al., 2014).” Minor Comments: In the background section of the abstract/methods, only one objective (attention and executive control) is mainly elaborated upon. I would also add the clinical objective, i.e., burnout, depression amelioration. Only QoL was briefly mentioned. Response: Since we did expect a good outcome on burnout and depression scores with the behavioral therapy, the main focus of the study was on the added value of tDCS in improving the more resilient attention deficits. Therefore, we primarily elaborated on that objective. We hope that we have made this clearer as follows in the abstract: Abstract: “Background: Burnout is characterized by deficiencies in attention and several components of the working memory. It has been shown that cognitive behavioral therapy can have a positive effect on burnout and depressive symptoms, however, the lingering effects of impaired attention and executive functions are the most frustrating.” The tDCS section of the introduction is not well structured. It’s better to subdivide the use of tDCS into neurological and psychiatric conditions. Now a distinction is made between “motor, cognitive, affective disorders” and “Alzheimer’s and MDD”. Response: Thank you for the thorough reading of the manuscript, this was indeed badly phrased. We have now changed it into the following, hopefully better, sentence: “tDCS is increasingly used in the treatment of motor, cognitive, and affective symptoms in different patient populations, both in neurological (e.g. Alzheimer’s disease; Flöel, 2014), and psychiatric disorders (e.g. major depressive disorder; Nitsche et al., 2009) (Brunoni et al., 2012).” Two papers on depression are mentioned: one submitting patients to 1 session and the other to 15 sessions. I would talk about the importance of the number of sessions as a potential variable that could increase tDCS effect (Brunoni)2 to explain why you submitted the patients to 15 sessions. Also, there are maybe one or two articles on stress in professionals, indirectly referring to burnout with no tDCS effect on burnout. One of the contributions of this article is that it may be the first looking at burnout. I would highlight this. Response: In the introduction, we have added an explanation for using repeated sessions instead of a single session in our study and we have pointed out that our study is probably the first to study the effects of tDCS in a burnout population. Thank you for this suggestion! “However, to induce a longer-lasting effect, repeated sessions are advised and it has already been shown that this can have a cumulative effect which is associated with greater magnitude and longer duration of the behavioral effects (Brunoni et al., 2012).The effects of tDCS have not yet been extensively evaluated in burnout patients. Some studies have used tDCS in stress-related patient populations, such as professional nurses (Stanton et al., 2015) or post-traumatic stress disorder (Saunders et al., 2015), however, to our knowledge, our study is the first to use tDCS in a burnout population.” I would also go more into details of other potential brain regions that could have been targeted, especially for those tasks that did not show a tDCS effect. Only DLPFC is mentioned. E.g. as mentioned above, inhibition has not been linked to DLPFC. Or why you included those tasks while targeting DLPFC. It could be the reason why no significance was observed in inhibition. Response: We have added several sentences to the discussion to clarify when we expected a result of left DLPFC stimulation and when it was rather unexpected. The discussion now reads as follows: “This study showed that three weeks of therapy, combined with real or sham stimulation, significantly improved several components of the central executive. However, analyses revealed that improvement was driven by a significant improvement after real tDCS for inhibition and shifting (WCST), and was also primarily seen after real tDCS for updating and control (TMT A, D2 G z, D2 G z – F). These results are in line with the study of Miler et al. who found that atDCS over the left DLPFC significantly improves the executive control of attention ( Miler et al., 2018). As in the study of Miler et al., no effect was seen on the percentage of mistakes, but the processing speed did improve more on the D2 test of attention after real tDCS than after sham tDCS ( Miler et al., 2018). However, the improvement on inhibition and shifting after tDCS was somewhat unexpected, since this is primarily associated with the right DLPFC and the right inferior frontal gyrus (Lie et al., 2006; Hampshire et al., 2010). Since this effect was only observed for the WCST, this might be related to this specific task. Indeed, studies have shown that, although the right DLPFC seems to be the most prominent in handling complex/manipulative working memory operations in the WCST (Lie et al., 2006), the left DLPFC is also involved during this task (Lie et al., 2006; Nagahama et al., 2005). atDCS also seemed to have a positive impact on other components of the working memory. The phonological loop might also be positively influenced by real tDCS as shown by a significant improvement of the real tDCS group on the BNT, although the sham group also trended towards a significant improvement. No effect was seen on the visuospatial sketchpad, which might not be surprising because this is believed to be situated primarily in the right prefrontal cortex (Suchan, 2008), but encoding clearly improved more after real atDCS than after sham tDCS (RBANS Immediate Memory Index). Transcranial magnetic stimulation (TMS) studies have shown a prominent role for the left DLPFC during encoding, observing shorter reaction times using a paired-pulse paradigm over this area (Gagnon et al., 2011). Though de Lara et al. (2017) did not find any effect of anodal tDCS over the left DLPFC on encoding, this might be due to the lesser intensity (1mA vs 2mA) they used in their study.” A paragraph on neurotransmitters could also be added. Specifically, alterations in dopamine and noradrenaline levels in DLPFC have been associated with impaired working memory performance. TMS and tDCS over DLPFC have also been shown to release dopamine in various brain regions, including the right ventral striatum. Further, tDCS to DLPFC has shown to improve participants’ memory accuracy, an effect that has been correlated significantly with dopamine release. Response: This might indeed be relevant information for the interested reader. We have added the following paragraph in the introduction: “One of the mechanisms that might be responsible for the cognitive problems in burnout patients is a dopaminergic dysfunction in the prefrontal cortex. It has been shown that dopamine in the prefrontal cortex plays a critical role in working memory and cognitive control (Polizzotto et al., 2020; Cools & D’Esposito, 2011) and that (chronic) stress can have a deteriorating effect on the dopaminergic system in this area (Mizoguchi et al., 2000). tDCS has been known to interact with dopaminergic systems (Polizzotto et al., 2020) and therefore tDCS over the DLPFC might be able to restore dopaminergic prefrontal cortex function.” In addition, a paragraph was added to the limitations section with the findings of a very recent paper on DLPFC stimulation and working memory: “Lastly, research has shown that the effect of tDCS on working memory might be dependent on, amongst others, the initial dopaminergic level that can impact the excitation/inhibition balance (i.e. homeostasis between relative contributions of excitatory and inhibitory synaptic inputs) (Polizzotti et al, 2020). More insight into the exact working mechanisms underlying the cognitive and attention deficits in burnout patients might be beneficial for future research.” I would also mention briefly why it took 3 years to recruit 15 patients. It could be helpful for future studies recruiting patients with burnout. Response: We have added some suggestions in the Limitations section to help with recruiting larger patient groups. As a single site with one therapist, it was very difficult to recruit several patients at the same time due to time constraints and a limited number of new intakes. In addition, several patients were discouraged by their primary physicians to participate in the experiment, since the primary physicians did not have enough information to judge the safety of tDCS. “Our study has several important limitations. First, our group of patients was relatively small. This is an important limitation given the positive trends of the effect of real tDCS on several outcome measures. Studies with more power will have to show whether these trends failed to reach significance due to a lack of power. In addition, some variables of interest (D2 s-score, TMT B, Stroop Card II, Raven) could not be interpreted correctly with the linear mixed model analysis because of a violation of assumptions. More data points could help to resolve this issue. Setting up multi-site cooperations to recruit participants and maintaining close relationships with primary care providers making them aware of the safety of tDCS when applied in the correct manner, could also help to convince patients to participate in tDCS studies. Larger groups to validate the efficacy of tDCS are crucial to investigate the clinical usability of this therapeutic aid.” Were the patients asked at the end of the treatment whether they knew they had received sham or real? (chi-squared test to test the integrity of masking).Were patients asked to evaluate their level of discomfort? The level of discomfort/pain has been associated with a TMS/tDCS effect. Response: Unfortunately, we did not systematically address these issues. We have added this to the limitations section and recommended it for future studies. “In addition, it is recommended to test for the efficiency of blinding the type of stimulation by asking the participants afterwards whether they think they were actively stimulated or not. Gathering information about the amount of discomfort could also be of importance for future studies using tDCS.”" } ] } ]	1	https://f1000research.com/articles/9-116
https://f1000research.com/articles/9-829/v1	30 Jul 20	{ "type": "Research Article", "title": "Impact of global warming on Raynaud’s phenomenon: a modelling study", "authors": [ "Charles Khouri", "Matthieu Roustit", "Jean-Luc Cracowski", "Matthieu Roustit", "Jean-Luc Cracowski" ], "abstract": "Background: Raynaud’s phenomenon is induced by excessive vasoconstriction of the peripheral microcirculation in response to environmental factors, essentially cold, but also stress or emotions. The objective of the present study is to evaluate the impact of global warming on the worldwide prevalence and severity of Raynaud’s phenomenon over the 21st century. Method: We first estimated the correlation between average temperature and prevalence and severity of Raynaud’s phenomenon. Then, we mapped the prevalence and the severity of Raynaud’s phenomenon worldwide at Christmas 1999 using historical data and, using climate projections from the Inter-Sectoral Impact Model Intercomparison Project, we predicted the prevalence and severity of Raynaud’s phenomenon at Christmas 2099 according to four greenhouse-gas emission scenarios. Results: The prevalence of Raynaud’s phenomenon in the general population is expected to decrease by 0.5% per degree Celsius increase. Furthermore, patients are expected to suffer from one less attack per week for each increase of 2.5 degrees Celsius. Conclusions: Our study shows that global warming may have a significant impact on the prevalence and the severity of Raynaud’s phenomenon over the 21st century. However, as expected, this will greatly depend on the level of greenhouse-gas emissions.", "keywords": [ "Raynaud's phenomenon", "global warming" ], "content": "\n\nRaynaud’s phenomenon is induced by excessive vasoconstriction of the peripheral microcirculation in response to environmental factors, essentially cold, but also stress or emotions1. Primary, or idiopathic, Raynaud’s phenomenon is the most frequent form (80–90%), while in some cases Raynaud’s phenomenon can be secondary to various auto-immune diseases (such as systemic sclerosis or systemic lupus erythematous) or drugs1. The prevalence of Raynaud‘s phenomenon is estimated to be approximatively 3 to 5% in the general population, with substantial variability according to climate and sex2. Exposure to cold increase sympathetic adrenergic outflow inducing cutaneous vasoconstriction by constricting skin toes and fingers arteriovenous anastomoses3. In individual with Raynaud’s phenomenon, the already-heightened sympathetic vasoconstriction is further amplified in intensity and will precipitate vasospasm of this vascular network2,3. Therefore sudden temperature change but also mean environmental temperature are determinant of Raynaud’s phenomenon burden and strong seasonal variation are described4–6. Most vasodilators currently used in Raynaud’s, such as nifedipine or sildenafil, only have limited efficacy, below the minimal clinically important difference7. Moreover, most recent trials have produced negative results, due to high heterogeneity and a significant placebo effect8.\n\nWe hypothesize that global warming should not leave Raynaud’s phenomenon as an unmet clinical need for too long. The objective of the present study is to evaluate the impact of global warming on the worldwide prevalence and severity of Raynaud’s phenomenon over the 21st century.\n\n\nMethod\n\nWe first estimated the correlation between average temperature and the prevalence of Raynaud’s phenomenon. The prevalence data were extracted from a systematic review of observational studies (Table 1)9. For each study we calculated the mean temperature during the winter preceding the publication of the study (from 1st November to 31 March) using historical climate data from the database developed by the Inter-Sectoral Impact Model Intercomparison Project (ISIMIP). The results were then extrapolated to other countries using latitudes coordinates.\n\nData is reproduced under the terms of the Creative Commons Attribution Non Commercial (CC BY-NC 4.0).\n\nWe further predicted the impact of global warming on the severity of Raynaud’s phenomenon, expressed as the average daily frequency of attacks, by using a model based on a Poisson regression including temperature (and other covariates), recently published by our team10 (this model is available online from DRYAD). This model is derived from a series of n-of-1 trials containing more than 2000 days of exposition, with daily temperature measurements collected at the nearest weather station to the patient’s home.\n\nFinally, we mapped the prevalence and the severity of Raynaud’s phenomenon worldwide at Christmas 1999 and, using climate projections from the ISIMIP, we predicted the prevalence and severity of Raynaud’s phenomenon at Christmas 2099, according to four greenhouse-gas emission scenarios (Representative Concentration Pathway (RCP) 2.6, RCP4.5, RCP6.0, and RCP8.5) described in the Fifth Assessment Report of the United Nations Intergovernmental Panel on Climate Change11. The HadGEM2-ES model was used for the modelling scenario12.\n\nThe RCPs represent the range of greenhouse-gas emission scenarios consistent with projections described in the literature; they include a mitigation scenario (RCP2.6), two intermediate scenarios (RCP4.5 and RCP6.0), and one scenario with high greenhouse-gas emissions (RCP8.5).\n\nData analysis were performed with R version 3.3.013 and map visualization with Panoply version 4.10.4 software14.\n\nA patient, Mrs Laurence Schuller, member of the board of the French Scleroderma Patient Association, was invited to comment on the study design and to interpret the results.\n\n\nResults\n\nWe found a high correlation between average temperature and the prevalence and severity of Raynaud’s phenomenon (p<0.001). According to these data, no Raynaud’s phenomenon attack is expected to occur above an average temperature of 13°C, which is consistent with individual data collected in our series of N-of-1 trials10. Consequently, the prevalence of Raynaud’s phenomenon in the general population is expected to decrease by 0.5% per degree Celsius increase. Furthermore, patients are expected to suffer from one less attack per week for each increase of 2.5 degrees Celsius.\n\nThe worldwide prevalence and severity of Raynaud’s phenomenon at Christmas 1999 and the range of predictions based on four greenhouse-gas emission scenarios at Christmas 2099 are shown in Figure 1.\n\n\nDiscussion\n\nOur study shows that global warming may have a significant impact on the prevalence and the severity of Raynaud’s phenomenon over the 21st century. However, as expected, this will greatly depend on the level of greenhouse-gas emissions. The most optimistic greenhouse gas scenario (RCP 2.6), which aims at keeping global warming below 2°C above pre-industrial temperatures, only has a limited impact on the global prevalence and severity of Raynaud’s phenomenon. On the other hand, scenarios without greenhouse-gas emission reductions (predictions ranging between RCP6.0 and RCP8.5) may largely improve the condition of patients suffering from Raynaud’s phenomenon. For example, people in western European countries could expect to be totally free of this painful and disabling condition in the event of the two higher gas-emission scenarios. Finally, patients in North America, Western Europe and Asia still suffering from Raynaud’s phenomenon are not expected to suffer more than one or two crises over the Christmas period in 2099.\n\nA limitation to our model is that we did not consider the potential increase in the use of air-conditioning and the stress caused by global warming, which may enhance RP. Climate change is also likely to result in temperature anomalies, including rapid temperature fluctuations which are known to be triggering factors of Raynaud’s phenomenon attacks. Nevertheless, mean temperatures are correlated to RP prevalence. In this study we only used one modelling scenario, the HadGEM2-ES model, which is widely used for climate research15,16, therefore uncertainty of our projections has not been evaluated but exist undoubtedly. The findings should thus be interpreted as potential impacts of climate change on Raynaud’s phenomenon according to one hypothetical scenario and not as projections.\n\n\nConclusion\n\nIn conclusion, this study shows that global warming is likely to have a significant impact on the prevalence and the severity of Raynaud’s phenomenon. Yet, whether the advantages of global warming will outweigh its drawbacks, even for Raynaud’s phenomenon patients, remains to be carefully scrutinized.\n\n\nData availability\n\nThe data from the N-of-1 trial PROFIL are freely available on datadryad.org\n\nDRYAD: Data from: On-demand sildenafil as a treatment for Raynaud phenomenon: a series of n-of-1 trials. https://doi.org/10.5061/dryad.c670tq217\n\nThe files required are:\n\n- PROFIL_DATA (The dataset of the study in plain text format with variables names as header 2306 observations on 50 variables)\n\n-model_1 (Final model)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\nHistorical and climate projections are available from the ISIMIP Earth System Grid Federation (ESGF) server in searching the climate forcing “HadGEM2-ES” and the variable “tasAdjust”:\n\n- tas_day_HadGEM2-ES_historical_r1i1p1_EWEMBI_landonly_19910101–20001231.nc (historical data)\n\n- tas_day_HadGEM2-ES_rcp26_r1i1p1_EWEMBI_landonly_20910101–21001231 (climate projection for RCP 2.6)\n\n- tas_day_HadGEM2-ES_rcp26_r1i1p1_EWEMBI_landonly_20910101–21001231 (climate projection for RCP 4.5)\n\n- tas_day_HadGEM2-ES_rcp26_r1i1p1_EWEMBI_landonly_20910101–21001231 (climate projection for RCP 6.0)\n\n- tas_day_HadGEM2-ES_rcp26_r1i1p1_EWEMBI_landonly_20910101–21001231 (climate projection for RCP 8.5)", "appendix": "Acknowledgement\n\nWe thank Mrs Laurence Schuller for her manuscript proofreading and comments.\n\n\nReferences\n\nHerrick AL: The pathogenesis, diagnosis and treatment of Raynaud phenomenon. Nat Rev Rheumatol. 2012; 8(8): 469–79. PubMed Abstract \| Publisher Full Text\n\nWigley FM, Flavahan NA: Raynaud’s Phenomenon. N Engl J Med. 2016; 375: 556–65. PubMed Abstract \| Publisher Full Text\n\nFlavahan NA: A vascular mechanistic approach to understanding Raynaud phenomenon. Nat Rev Rheumatol. 2015; 11(3): 146–58. PubMed Abstract \| Publisher Full Text\n\nWatson HR, Robb R, Belcher G, et al.: Seasonal variation of Raynaud’s phenomenon secondary to systemic sclerosis. J Rheumatol. 1999; 26(8): 1734–7. PubMed Abstract\n\nPauling JD, Reilly E, Smith T, et al.: Factors Influencing Raynaud Condition Score Diary Outcomes in Systemic Sclerosis. J Rheumatol. 2019; 46(10): 1326–34. PubMed Abstract \| Publisher Full Text\n\nHughes M: Effect of Season on Internet Searches for Information on Raynaud Phenomenon. J Rheumatol. 2019; 46(11): 1543–4. PubMed Abstract \| Publisher Full Text\n\nKhouri C, Lepelley M, Bailly S, et al.: Comparative efficacy and safety of treatments for secondary Raynaud’s phenomenon: a systematic review and network meta-analysis of randomised trials. Lancet Rheumatol. 2019; 1(4): e237–46. Publisher Full Text\n\nSeibold JR, Wigley FM: Editorial: Clinical Trials in Raynaud’s Phenomenon: A Spoonful of Sugar (Pill) Makes the Medicine Go Down (in Flames). Arthritis Rheumatol. 2017; 69(12): 2256–8. PubMed Abstract \| Publisher Full Text\n\nGarner R, Kumari R, Lanyon P, et al.: Prevalence, risk factors and associations of primary Raynaud’s phenomenon: systematic review and meta-analysis of observational studies. BMJ Open. 2015; 5(3): e006389. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoustit M, Giai J, Gaget O, et al.: On-Demand Sildenafil as a Treatment for Raynaud Phenomenon: A Series of n-of-1 Trials. Ann Intern Med. 2018; 169(10): 694–703. PubMed Abstract \| Publisher Full Text\n\nIPCC: Climate Change 2014: Synthesis Report. Contribution of Working Groups I,II and III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change [Core Writing Team, R.K. Pachauri and L.A. Meyer (eds.)]. IPCC, Geneva, Switzerland, 2014; 151. Reference Source\n\nCollins WJN, Bellouin N, Doutriaux-Boucher M, et al.: Evaluation of the HadGEM2 model. Met Office Hadley Centre Technical Note no. HCTN 74 available from Met Office, FitzRoy Road, Exeter EX1 3PB. 2008. Reference Source\n\nR Core Team: R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. 2016. Reference Source\n\nNASA GISS: Panoply 4 netCDF, HDF and GRIB Data Viewer. (accessed 1 Jul 2019). Reference Source\n\nWarszawski L, Frieler K, Huber V, et al.: The Inter-Sectoral Impact Model Intercomparison Project (ISI-MIP): Project framework. Proc Natl Acad Sci U S A. 2014; 111(9): 3228–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuo Y, Gasparrini A, Li S, et al.: Quantifying excess deaths related to heatwaves under climate change scenarios: A multicountry time series modelling study. PLoS Med. 2018; 15(7): e1002629. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoustit M, Giai J, Gaget O, et al.: Data from: On-demand sildenafil as a treatment for Raynaud phenomenon: a series of n-of-1 trials. Dryad, Dataset, 2019. http://www.doi.org/10.5061/dryad.c670tq2" }	[ { "id": "73786", "date": "09 Nov 2020", "name": "Rossella De Angelis", "expertise": [ "Reviewer Expertise Rheumatology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe predicted prevalence and the severity of Raynaud's phenomenon at Christmas 2099, according to four greenhouse gas emission scenarios as described in the 5th Assessment Report of United Nations. So:\nIf possible, a slightly detailed explanation of reference item 11 would be particularly welcome, also to make the data more understandable to doctors.\n\nA rather more refined description of the statistical model HadGe2_ES (not only with a simple bibliographic citation), in order to make the reading of the work more fluent. On the other hand, a doctor who is interested in the study should go into the bibliographic item no. 12, which is not easily understandable at a first reading.\nThe work is, however, very interesting and describes a likely scenario. Those clinicians who deal with Raynaud's phenomenon should take this hypothesis into account. In any case, a welcome reading.\n\nIs the work 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": [] }, { "id": "86792", "date": "22 Jun 2021", "name": "Rebecca S. Overbury", "expertise": [ "Reviewer Expertise Rheumatology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a research article that attempts to hypothetically model how rising temperatures, assumed due to increased levels of greenhouse gas emissions, would affect the prevalence and severity (as defined by frequency of \"crises\") of Raynaud's phenomenon. Overall, this is a truly fascinating, if disturbing, question to consider and one that is likely to be relevant for worldwide clinicians moving forward. The authors present previous research supporting a sound hypothesis from which to ask this question. I find this a novel and interesting research question.\n\nI do not have expertise to speak to the methods of modelling in particular. However, the authors are transparent, if succinct, in explaining their methods and all necessary materials for reproducibility appear to be available. Someone well-versed in this may desire more detail.\nAs a Rheumatologist, I recognize that the affect of this climate change phenomenon in patients with primary versus secondary Raynaud's is likely to differ; and I would be interested to hear more about this. This is not addressed here, but is arguably outside the scope of this methodology.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-829
https://f1000research.com/articles/9-793/v1	29 Jul 20	{ "type": "Research Article", "title": "Cost analysis of implementing HIV drug resistance testing in Kenya: a case study of a service delivery site at a tertiary level hospital in Kenya", "authors": [ "Rachael W. Gachogo", "Daniel N. Mwai", "Frank G. Onyambu", "Daniel N. Mwai", "Frank G. Onyambu" ], "abstract": "Background: HIV drug resistance (HIVDR) threatens progress achieved in response to the HIV epidemic. Understanding the costs of implementing HIVDR testing programs for patient management and surveillance in resource-limited settings is critical in optimizing resource allocation. Here, we estimate the unit cost of HIVDR testing and identify major cost drivers while documenting challenges and lessons learnt in implementation of HIVDR testing at a tertiary level hospital in Kenya. Methods: We employed a mixed costing approach to estimate the costs associated with performing a HIVDR test from the provider’s perspective. Data collection involved a time and motion study of laboratory procedures and interviewing laboratory personnel and the management personnel. Cost analysis was based on estimated 1000 HIVDR tests per year. Data entry and analysis were done using Microsoft Excel and costs converted to US dollars (2019). Results: The estimated unit cost for a HIVDR test was $271.78 per test. The main cost drivers included capital ($102.42, 37.68%) and reagents (101.50, 37.35%). Other costs included: personnel ($46.81, 17.22%), utilities ($14.69, 5.41%), equipment maintenance costs ($2.37, 0.87%) and quality assurance program ($4, 1.47%). Costs in relation to specific laboratory processes were as follows: sample collection ($2.41, 0.89%), RNA extraction ($22.79, 8.38%), amplification ($56.14, 20.66%), gel electrophoresis ($10.34, 3.80%), sequencing ($160.94, 59.22%), and sequence analysis ($19.16, 7.05%). A user-initiated modification of halving reagent volumes for some laboratory processes (amplification and sequencing) reduced the unit cost for a HIVDR test to $233.81 (13.97%) reduction. Conclusions: Capital expenditure and reagents remain the most expensive components of HIVDR testing. This cost is bound to change as the sequencing platform is utilized towards maximum capacity or leveraged for use with other tests. Cost saving in offering HIVDR testing services is also possible through reagent volume reduction without compromising on the quality of test results.", "keywords": [ "HIV", "HIV drug resistance testing", "implementation science", "cost analysis", "health systems strengthening" ], "content": "Background\n\nUnprecedented increased access to antiretroviral therapy (ART) is one of the greatest milestones in the fight against the HIV epidemic, resulting in reduced mortality from AIDs-related causes and a global decline in HIV incidence (UNAIDS, 2019). However, this success is threatened by emergence of HIV drug resistance (HIVDR). The World Health Organization (WHO) reports greater than 10% pretreatment drug resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) among adult patients starting on a first-line ART regimen. This rate is higher in children below 18 months, with over half of newly diagnosed infants harboring resistance to NNRTIs. The prevalence of acquired HIVDR among patients on ART ranges from 3% to 29% (WHO, 2019). Moreover, recent studies in Kenya have shown an upward trend in both transmitted and acquired HIVDR (Hassan et al., 2019; Kantor et al., 2018; Milne et al., 2019).\n\nART is delivered through the public health approach in most low- and middle-income countries, where standardized drug regimens are administered with simplified laboratory monitoring using tests such as HIV viral load and CD4 count assays (Lessells et al., 2013; De Luca et al., 2013; Phillips et al., 2018). Access to HIVDR testing is limited for patients in resource-limited settings such as Kenya due to high costs involved and inadequate laboratory capacity (Kennedy et al., 2016; Nkengasong et al., 2018; Petti et al., 2006). On the other hand, HIVDR testing is offered routinely in resource-rich setting to inform clinical management of people living with HIV (Dunn et al., 2011; Günthard et al., 2019). However, considerable effort has been made to monitor population level of HIVDR in low- and middle-income countries by implementing HIVDR surveys according to WHO guidelines. These surveys have been crucial in informing national ART guidelines; for example, data on the high prevalence of pretreatment drug resistance to NNRTIs has been critical in the transitioning from an NNRTI-based first-line regimen to a regimen that consists of dolutegravir (DTG) in sub-Saharan countries (WHO, 2019).\n\nFunding for HIV programming in Sub-Saharan countries is provided by multilateral development partners including the US President’s Emergency Plan for AIDs Relief (PEPFAR), the Global Fund and the World Bank, among others (Ministry of Health, 2015; Mwai, 2017; Olakunde et al., 2019; Schneider et al., 2016). These multilateral partners have acknowledged the integral role played by quality medical laboratory services within health systems, precipitating mobilization of significant amounts of funding earmarked for laboratory systems strengthening in resource-limited settings (Abimiku, 2009; Hamel et al., 2015; Nkengasong et al., 2018). This support has led to great improvement in HIV diagnostic and monitoring services, inter-laboratory networks, systems, governance, and institutions, evident by the increased number of laboratories that have the capacity to perform molecular diagnostics for HIV viral load and early infant diagnosis (EID), as well as HIV genotyping. Furthermore, these partners have been at the forefront in supporting development of national laboratory policy and strategic plans, and improving quality in these laboratories (Nkengasong et al., 2018; Hamelet al., 2015).\n\nIn Kenya, there are 10 laboratories across the country that support HIV viral load and EID testing through a PEPFAR and Global Fund funded specimen referral network, and only four of these have capacity to perform HIVDR testing. However, there is a paucity of costing data from these laboratories and no detailed cost analysis has been done (Inzaule et al., 2013). Some of the essential costs when performing a costing analysis include equipment costs, personnel costs, and utilities costs. Previous studies have included reagents cost and consumables cost in their estimations, excluding major cost categories (Acharya et al., 2014; Inzaule et al., 2013; Novitsky et al., 2015; Zhou et al., 2011). A detailed cost analysis provides a deeper understanding of the costs of HIVDR to the health systems. Moreover, cost information is important in development of business plans, projections, planning, budgeting, pricing and resources allocation. Finally, it gives a good guide on the affordability of HIVDR testing inclusion in the standard package of HIV testing in Kenya to alleviate the rising number of cases of HIVDR.\n\nIn this study we estimate the unit cost of HIVDR testing, identify the cost drivers for the HIVDR test, explore opportunities for cost saving and document challenges and lessons learnt in implementation of HIVDR testing.\n\n\nMethods\n\nEthical approval was obtained from University of Nairobi/Kenyatta National Ethics Review Committee (KNH-UON-ERC- P562/01/2019). Verbal informed consent was obtained from all participants for the interviews and the ethics review board waived the need for a signed consent form. the study followed the guidelines for verbal consent, including explaining to the study participants the pertinent issues about the study including the purpose, benefits, risks and procedures. The participants were also given enough time to decide whether to participate or not as well as an opportunity to ask questions.\n\nThis costing study was conducted at the Molecular and Infectious Diseases Research Laboratory (MIDRL) located within the Kenyatta National Hospital and University of Nairobi School of Medicine. The MIDRL supports the implementation of high quality, sustainable and comprehensive HIV prevention, care and treatment in Nairobi through provision of laboratory services including HIV viral load, HIV early infant diagnosis and HIVDR testing. Data collection was performed during the initial implementation stages, that is within the first year of commencing HIVDR testing.\n\nThe study utilized both micro and gross costing in quantification and valuation of the cost categories, which was done from the provider’s perspective.\n\nData were collected and compiled from 1st January to 30th June 2019. Costs were assessed for various processes in the HIVDR testing workflow including laboratory administration, sample collection and preparation, viral RNA extraction, nucleic acid amplification, gel electrophoresis, Sanger sequencing, data analysis and reporting. At the time of data collection, the laboratory used a commercial HIVDR assay supplied by Thermofisher (Cat. no. 12183018A, Waltham, USA). Cost data were collected on annualized depreciation for capital items including laboratory equipment and furniture, long term training and information technology equipment at a depreciation rate of 10%, reagents and consumables, personnel, utilities, laboratory and office space, quality assurance program, and maintenance costs. Cost details were obtained from quotations, invoices and delivery notes.\n\nData was collected by RG, who at the time of the study was working at MDRL as a clinical laboratory assistant and a master’s student (Health Economics and Policy) at the University of Nairobi. Data collection involved a time and motion study of the laboratory procedures for HIVDR testing and interviewing of laboratory and management personnel. The time and motion study was carried out in 12 sessions lasting between 1–3 hours based on the length of the laboratory procedure. A structured questionnaire depicting all the HIVDR testing steps and data collection tables were used to document quantity of reagents and consumables used as well as duration of each HIVDR process (Gachogo et al., 2020b). Two laboratory technical staff and one member of management were interviewed in three sessions (1 hour) each. Individuals with in-depth knowledge on HIVDR testing implementation were purposively selected to participate in the interviews. Interviews took place within their work environment. An interview guide was used to conduct the interview process and the data was recorded in form of field notes (Gachogo et al., 2020b). Technical staff were interviewed about HIVDR testing processes and experiences of implementing testing, while the member of management was interviewed about cost data, which was recorded in the data collection tables. None of the interviewees declined to participate. Administrative records such as invoices, requests for quotations and delivery notes obtained from the program archives were reviewed to obtain purchase costs. In addition, the laboratory personnel were asked to narrate their experience in setting up a HIVDR testing laboratory, with particular interest on problems encountered and solutions to these set-backs.\n\nWe developed a Microsoft Excel version 2010 based model to aid in estimation of the unit cost and cost for the various categories and laboratory processes. All costs were converted to US dollars (13th April 2019, $1 USD = 101.2 Kshs). The MIDRL projected to perform 1000 tests in 2019 based on the number of HIVDR tests performed in the first and second quarter of the 2019 financial year. Responses on challenges experienced during the implementation of HIVDR testing were manually scanned through by RG and FO for developing themes and coded accordingly.\n\nOne-way sensitivity analysis for 20% variations to cost categories was performed to establish the level of uncertainty linked with costs variation of inputs to HIVDR test. This involved varying capital, personnel, reagents, maintenance, and quality assurance program costs by ±20% and evaluating how each of them influence the HIVDR unit cost relative to the estimated cost.\n\n\nResults\n\nHIVDR testing is carried out in five major processes; namely, sample collection and preparation, nucleic acid extraction, nucleic acid amplification, sequencing and sequence analysis. Specimen collection and preparation involves collection of whole blood from the patient into blood collection tubes that contain ethylenediaminetetraacetic acid (EDTA) anticoagulant. Once the blood is collected into the blood collection tubes, the specimen is prepared for storage by spinning, pipetting and aliquoting into storage vials. The second step in HIV resistance testing is nucleic acid extraction from the plasma. In this step, HIV ribonucleic acid (RNA) is isolated from plasma. Once extracted and purified, the nucleic acid is converted to complementary deoxy-ribonucleic acid (cDNA) and amplified by polymerase chain reaction (PCR) and sequenced. Sequencing involves amplicon purification, cycle sequencing, amplicon purification, sequence detection and visualization. The last step in HIVDR testing is sequence analysis, which involves sequence data validation, sequence assembly, interpretation and quality analysis.\n\nActivity-based costing for HIVDR testing was performed at MIDR Laboratory by collecting cost data for each step in the drug resistance testing. The cost for performing HIVDR testing was US$ 271.78 per test, where capital costs took the biggest share at $102.42, followed by reagents and consumables at $101.5 (Table 1 and Figure 1). Other costs included personnel ($46.81), utilities ($14.69), maintenance cost of equipment ($2.37) and quality assurance program ($4.00) (Gachogo et al., 2020a).\n\nCosts in USD.\n\n* The most costly component of HIV resistance testing.\n\nUnit cost is $271.8.\n\nThe sequencing step had the largest cost of $160.94 per test, while DNA/RNA amplification had the second largest cost of $56.14. DNA/RNA extraction, gel electrophoresis, sequence analysis and sample collection had a cost of $22.79, $10.34, $19.16 and $2.41, respectively (Table 2 and Figure 2).\n\nCosts in USD.\n\n* The most expensive step in HIV drug resistance testing.\n\nUnit cost is $271.78.\n\nThe laboratory validated a low-cost assay, whereby reagents volumes used during the amplification and sequencing steps were half the recommended volumes by the manufacturer. The test performance was in agreement with the original assay as previously reported (Magomere et al., 2019). The unit cost as a result of halving reagents volumes at the amplification and sequencing step was $233.81, a reduction from $271.78 of the original assay. There was a notable reduction for the amplification and sequencing costs to $38.38 and $140.70 from $56.14 and $160.94, respectively. There was no change in costs for the other steps in HIVDR testing (Table 3 and Figure 3).\n\n1 Halving reagent volumes at DNA/RNA amplification step reduces the cost of this step from $56.14 to $38.38.\n\n2 Halving reagent volumes at sequencing step reduces the cost of this step from $160.94 to 140.70.\n\n3 Halving reagent volumes at DNA/RNA amplification and sequencing steps reduce the HIV drug resistance test cost to $ 233.81 from $ 271.78.\n\nTotal unit cost is $233.81.\n\nThe cost for the HIVDR test using Viroseq HIV genotyping reagents and consumables (Abbott Molecular, Abbott Park, IL) was estimated at $379.46. This is one of the FDA-approved HIVDR tests available on the market and is used as an alternative to in-house reagents and consumables manufactured by Thermofisher. Reagents and consumables accounted for 55% ($209.18) of the unit cost of the HIVDR testing (Table 4).\n\n* Reagents and consumables are the most expensive inputs to HIV drug resistance testing in Viroseq HIV genotyping.\n\nAs a startup laboratory, the challenges and lessons learnt during the processes of establishing such a capital-intensive undertaking in a resource-limited setting were documented. Table 5 shows some of the challenges and lessons learnt.\n\nThe costs presented assume that the laboratory runs 1000 HIVDR tests per year with no machine breakdown or waste of supplies. Considering that variation in input costs would have an impact on the input costs, a one-way sensitivity analysis for 20% variations to cost categories was performed. Variations to capital, reagents, and personnel inputs had a major impact on the unit cost, whereas variations to utilities, maintenance and quality assurance results had no significant impact on the unit cost. A 20% variation to capital and reagents results in changes of up to 7.5% in unit cost; approximately a $20 difference (Figure 4).\n\nGrey represents 20% reduction in the input costs, while dark grey represents 20% increase in the input costs. Unit cost is $271.78.\n\n\nDiscussion\n\nThe aim of this study was to establish a detailed cost profile for HIVDR testing from a provider’s perspective and identify cost drivers. We also report the challenges encountered and lessons learnt during the implementation of HIVDR testing at the MIDRL. The cost of performing HIVDR testing was estimated to be $271.78 per test. The cost estimate represents all the inputs required for performing HIVDR testing including; capital, personnel, reagents, consumables, quality assurance program and service contracts for the laboratory equipment for performing 1000 tests per year. Previous studies did not include all cost categories, making it difficult to compare costs for offering drug resistant tests across many laboratories (Acharya et al., 2014; Alemán et al., 2015; Inzaule et al., 2013; Novitsky et al., 2015). In this study, we offer a framework for performing laboratory cost analyses that makes it easy to compare cost categories between different laboratories. A previous study from KEMRI/CDC, Kenya, performed a cost analysis of their in-house assay and established the unit cost to be approximately $113.33, with $109.31 as the cost of reagents and consumables (Inzaule et al., 2013). The analysis included the costs of reagents and consumables and the cost of maintaining the equipment but did not account for capital, personnel and external quality assurance program costs. Considering the reagent and consumable costs, there was a correlation in the cost results for these items, as our study estimated the cost to be $101.50. The slight difference could be attributed to time difference in performing the cost analysis. In addition, the previous study estimated the equipment maintenance cost to be $4.02, while the present study estimates a cost of $2.37 for this category. Some of the studies performed in other parts of the world found considerably lower reagents and consumables costs than those estimated by this study. For instance, the costs reported in India and Cuba were $85.00 and $87.80, respectively (Acharya et al., 2014; Alemán et al., 2015). Conversely, one study reported higher reagent costs than those found in the present study; the estimated cost was $139.75 per test (Novitsky et al., 2015).\n\nTo answer the question of the cost drivers for HIVDR testing, the costs were categorized according to the processes involved in HIVDR testing, including sample collection, RNA extraction and amplification, gel electrophoresis, sequencing, and sequencing analysis. In terms of cost categories, capital cost took the biggest share of pie at $102.42 (37.68%), followed by reagents plus consumables at $101.50 (37.35%). High capital costs could be attributed to sub-optimal utilization of the sequencing platform. It should be noted that the equipment required for HIVDR testing can be leveraged to perform more patient tests that support HIV care and treatment, therefore bringing down the cost of equipment attributed to HIVDR testing. Our cost analysis was based on an estimated projection of 1000 HIVDR tests per year, which is a gross underestimation of the laboratory’s capacity. If the laboratory operated optimally, offering approximately ~6720 tests per year, the capital cost would reduce ~6.7 fold. The reagent costs were considerably high as a result of acquiring the sequencing machine at no upfront cost. This bound the laboratory to only procure reagents and consumables from the machine provider. This commitment denies the laboratory an opportunity to practice strategic purchasing, which would be a key factor in lowering the cost of reagents. This is not unique to HIVDR testing; a study done in Kenya to estimate cost of HIV viral load and EID reported high reagent costs as a result of machine acquisition on a placement basis (Cintron et al., 2017).\n\nA comparison with other studies was impossible as most of the previous cost analysis included reagents and consumables costs only, omitting other categories such as capital, personnel, utilities and quality control program costs (Alemán et al., 2015; Inzaule et al., 2013; Novitsky et al., 2015). In terms of cost per process, the sequencing step, which involves purification of PCR products, cycle sequencing, purification of sequencing products and sequence detection, was the most costly step in HIVDR testing at $160.94 (59.22%). This is in keeping with other studies evaluating the cost of HIVDR testing. Other studies report $59.88 (52.92%) and $50 (58.82%) as the cost for the sequencing step (Acharya et al., 2014; Inzaule et al., 2013). The one-way sensitivity analysis performed illustrates a cost saving opportunity, for example through negotiating lower reagent prices and maximizing utilization of the sequencing platform, therefore ensuring sustainable use of health financing resources.\n\nComparing the cost of the HIVDR test to a HIV viral load test used in monitoring and management of people living with HIV, the HIVDR testing cost is higher. A study in Kenya estimates HIV viral load test at $24.63 for non-point-of-care viral load testing and $29.74 for point-of-care HIV viral load testing (Cintron et al., 2017). This is attributed to additional processes in HIVDR test, that is, nested PCR and cycle sequencing processes. These increase the amount of cost inputs used in HIVDR testing, especially staff hands-on time.\n\nThe study evaluated the effects of reducing the reagents volume on the cost and performance characteristics of the HIVDR testing in view of reagents being one of the cost drivers for HIVDR testing. On the cost of the HIVDR testing, there was a significant reduction in the cost from $271.78 to $247.30; a ~13.97% reduction in the cost per test. This assay modification led to a ~37.68% reduction in reagent costs. Of note is the concordance of the two assays in their performance characteristics, which increases the confidence in adoption of this cost saving undertaking by the laboratories that would like to increase their efficiency in offering the HIVDR testing service (Magomere et al., 2019). The new assay performance characteristics met the WHO HIVDR validation criteria (WHO, 2018). Cost computation using Viroseq reagents, which are FDA approved and an alternative to in-house Thermofisher (Illinois, US) reagents, gave a cost of $379.46 per test, with reagents taking the biggest share of the cost at 55.13% ($209.18). This illustrates a lower cost of HIVDR testing using Thermofisher reagents by $107.68. These findings are in keeping with other studies where the cost of HIVDR per test was lower when using the in-house system compared to the Viroseq system (Acharya et al., 2014; Inzaule et al., 2013; Zhou et al., 2011). For instance, one study reported a $132.86 difference in the two systems, while another reported a $165.01 difference (Inzaule et al., 2013).\n\nOne of the challenges encountered during the implementation of HIVDR testing was high staff turnover. This is attributed to advanced molecular skills required for sample analysis in HIVDR testing. There are a few laboratory specialists equipped with these skills, making them highly sought after in the job market. This is a challenge in a low resource set up as training personnel on this area is quite expensive (Abimiku, 2009; Kennedy et al., 2016; Nkengasong et al., 2018; WHO, 2010). To counteract this challenge, one of the staff won a training grant to learn HIVDR testing from a laboratory that was already established. The sequencing machine provider is also bound by the contract to train the laboratory staff to the highest level possible and provide machine service when due. Maintaining a good working relationship with other laboratories performing the test helps in the exchange of new ideas and also facilitates an inter-laboratory proficiency testing program.\n\nUnlike HIV viral load and EID, HIVDR testing is not included in the Global Access Program, which has helped in the scaling up of HIV viral load and EID testing in Kenya at a relatively low cost (WHO, 2014). This raises sustainability concerns owing to recent reduced donor funding for HIV programs. However, HIVDR testing services can leverage on already established sample referral networks, human resources, laboratory equipment and database for HIV viral load. The multiple possible applications of the sequencing platform provides opportunities to deploy it for other tests and services, therefore reducing the overall running costs. Sensitization of key stakeholders involved in management of people living with HIV through regular stakeholders meetings has been instrumental in uptake of HIVDR test.\n\nOther challenges experienced during the implementation of drug resistance testing included frequent electricity disconnections, which was solved by installing a backup generator to ensure a constant supply of power. This corroborates other previous studies that highlighted similar findings in resource limited settings (Kennedy et al., 2016; Nkengasong et al., 2018). Furthermore, supply chain insufficiency, which delayed timely delivery of reagents, consumables, and laboratory equipment, was a major setback in implementing HIVDR testing. Finally, the premixed PCR master-mixes limited the flexibility of their use for other tests.\n\n\nStrengths of the study\n\nThis report presents findings from a complete cost analysis performed in the early stages of implementation of HIVDR testing, hence giving a good picture of the costs involved in the process. This report will further serve as a useful resource for planning and budgeting information for better resource management for similar projects in future. The inclusion of the cost-saving assay evaluation makes the study one of a kind, as it provides an evidence of cost reduction and comparable performance characteristics for both assays.\n\n\nStudy limitations\n\nThe study estimated costs from the provider’s perspective, thus limiting the inclusion of cost incurred by patients. The study design also excluded transport costs incurred for the transport of samples from peripheral health facilities to the testing laboratory in Nairobi. Furthermore, the cost analysis was carried out in only one facility, hence hindering the comparison across facilities offering HIVDR testing. This study presents a partial economic evaluation; a complete economic evaluation would give a clearer picture on the cost-effectiveness of HIVDR testing versus the status quo. Finally, at the time of the study the laboratory was not operating at full capacity, which increases the unit cost of HIVDR testing. It is conceivable that once uptake for the HIVDR test increases, the additional volumes would translate to reduced costs.\n\n\nConclusion\n\nThe MIDRL has implemented HIVDR testing capacity for patients failing ART at a cost of $271.78 per test. The most important cost driver is expenditure on capital cost, which is likely to reduce when utilization of the equipment increases. It has also been demonstrated that there are opportunities for cost saving through assay modifications such as selective reagent volume reduction.\n\n\nData availability\n\nFigshare: Cost analysis of implementing HIV drug resistance testing in Kenya: a case study of a service delivery site at a tertiary level hospital in Kenya: https://doi.org/10.6084/m9.figshare.12561980.v3 (Gachogo et al., 2020a).\n\nThis project contains the following underlying data:\n\n- HIVDR_Consumables cost.xlsx\n\n- HIVDR_BuildingCost.xlsx\n\n- HIVDR_Electricitycost.xlsx\n\n- HIVDR_Equipment_cost.xlsx\n\n- HIVDR_Indirectcost.xlsx\n\n- HIVDR_Personnel_cost.xlsx\n\n- HIVDR_Reagents_cost.xlsx\n\n- HIDVR_Viroseq_consumbles.xlsx\n\n- HIDVR_viroseq_reagents.xlsx\n\n- HIVDR_Field_notes.pdf\n\nFigshare: Cost analysis of implementing HIV drug resistance testing in Kenya: a case study of a service delivery site at a tertiary level hospital in Kenya. https://doi.org/10.6084/m9.figshare.12628031.v1 (Gachogo et al., 2020b).\n\n- HIVDR_Questionnaire.pdf\n\n- Interview Guide.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\nAbimiku AG; Institute of Human Virology, University of Maryland School of Medicine PEPFAR Program (AIDS Care Treatment in Nigeria [ACTION]): Building laboratory infrastructure to support scale-up of HIV/AIDS treatment, care, and prevention: In-country experience. Am J Clin Pathol. 2009; 131(6): 875–886. PubMed Abstract \| Publisher Full Text\n\nAcharya A, Vaniawala S, Shah P, et al.: Development, Validation and Clinical Evaluation of a Low Cost In-House HIV-1 Drug Resistance Genotyping Assay for Indian Patients. PLoS One. 2014; 9(8): e105790. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlemán Y, Vinken L, Kourí V, et al.: Performance of an in-house human immunodeficiency virus type 1 genotyping system for assessment of drug resistance in Cuba . PLoS One. 2015; 10(2): e0117176. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCintron C, Mudhune V, Haider R, et al.: Costs of Hiv Viral Load and Early Infant Diagnosis Testing in Kenya. 2017. Reference Source\n\nDe Luca A, Hamers RL, Schapiro JM: Antiretroviral Treatment Sequencing Strategies to Overcome HIV Type 1 Drug Resistance in Adolescents and Adults in Low-Middle-Income Countries. J Infect Dis. 2013; 207(Suppl 2): S63–9. PubMed Abstract \| Publisher Full Text\n\nDunn DT, Coughlin K, Cane PA: Genotypic resistance testing in routine clinical care. Curr Opin HIV AIDS. 2011; 6(4): 251–257. PubMed Abstract \| Publisher Full Text\n\nGachogo R, Mwai D, Onyambu F: Cost analysis of implementing HIV drug resistance testing in Kenya: a case study of a service delivery site at a tertiary level hospital in Kenya. figshare. Dataset. 2020a. http://www.doi.org/10.6084/m9.figshare.12561980.v3\n\nGachogo R, Mwai D, Onyambu F: Cost analysis of implementing HIV drug resistance testing in Kenya: a case study of a service delivery site at a tertiary level hospital in Kenya. figshare. Dataset. 2020b. http://www.doi.org/10.6084/m9.figshare.12628031.v1\n\nGünthard HF, Calvez V, Paredes R, et al.: Human Immunodeficiency Virus Drug Resistance: 2018 Recommendations of the International Antiviral Society - USA Panel. Clin Infect Dis. 2019; 68(2): 177–187. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHamel DJ, Sankalé JL, Samuels JO, et al.: Building laboratory capacity to support HIV care in Nigeria: Harvard / APIN PEPFAR, 2004–2012. Afr J Lab Med. 2015; 4(1): 190. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHassan AS, Bibby DF, Mwaringa SM, et al.: Presence, persistence and effects of pre-treatment HIV-1 drug resistance variants detected using next generation sequencing: A Retrospective longitudinal study from rural coastal Kenya. PLoS One. 2019; 14(2): e0210559. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInzaule S, Yang C, Kasembeli A, et al.: Field Evaluation of a Broadly Sensitive HIV-1 In-House Genotyping Assay for Use with both Plasma and Dried Blood Spot Specimens in a Resource-Limited Country. J Clin Microbiol. 2013; 51(2): 529–539. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKantor R, Delong A, Schreier L, et al.: HIV-1 second-line failure and drug resistance at high-level and low-level viremia in Western Kenya. AIDS. 2018; 32(17): 2485–2496. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKennedy SB, Dogba JB, Wasunna CL, et al.: Pre-Ebola virus disease laboratory system and related challenges in Liberia. Afr J Lab Med. 2016; 5(3): 508. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLessells RJ, Avalos A, de Oliveira T: Implementing HIV-1 Genotypic Resistance Testing in Antiretroviral Therapy Programs in Africa: Needs, Opportunities, and Challenges. AIDS Rev. 2013; 15(4): 221–229. PubMed Abstract \| Free Full Text\n\nMagomere EO, Nyangahu DD, Kimoloi S, et al.: Performance characteristics of a modified HIV-1 drug resistance genotyping method for use in resource-limited settings [version 1; peer review: 2 approved]. F1000Res. 2019; 8: 15181. Publisher Full Text\n\nMilne RS, Silverman RA, Beck IA, et al.: Minority and majority pretreatment HIV-1 drug resistance associated with failure of first-line nonnucleoside reverse-transcriptase inhibitor antiretroviral therapy in Kenyan women. AIDS. 2019; 33(6): 941–951. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMinistry of Health: Kenya Nationl health accounts 2012/2013. 2015. Reference Source\n\nMwai D: Kenya National Health Accounts FY 2015/16 Republic of Kenya Ministry of Health. 2017. Publisher Full Text\n\nNkengasong JN, Peeling RW, Yao K, et al.: Personal View Laboratory medicine in Africa since 2008 : then , now , and the future. Lancet Infect Dis. 2018; 3099(18): 1–6. Publisher Full Text\n\nNovitsky V, Zahralban-steele M, Mclane F: Long-Range HIV Genotyping Using Viral RNA and Proviral DNA for Analysis of HIV Drug Resistance and HIV Clustering. J Clin Microbiol. 2015; 53(8): 2581–2593. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOlakunde BO, Adeyinka DA, Ozigbu CE, et al.: Revisiting aid dependency for HIV programs in Sub-Saharan Africa. Public Health. 2019; 170: 57–60. PubMed Abstract \| Publisher Full Text\n\nPetti CA, Polage CR, Quinn TC, et al.: Laboratory Medicine in Africa: A Barrier to Effective Health Care. Clin Infect Dis. 2006; 42(3): 377–382. PubMed Abstract \| Publisher Full Text\n\nPhillips AN, Cambiano V, Nakagawa F, et al.: Cost-effectiveness of public-health policy options in the presence of pretreatment NNRTI drug resistance in sub-Saharan Africa: a modelling study. Lancet HIV. 2018; 5(3): e146–e154. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchneider MT, Birger M, Haakenstad A, et al.: Tracking development assistance for HIV/AIDS: the international response to a global epidemic. AIDs. 2016; 30: 1475–1479. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUNAIDS: UNAIDS DATA 2019. 2019. Reference Source\n\nWHO: Recommended Methods for Validation of an In-House Genotyping Assay for Surveillance of HIV Drug Resistance. 2018.\n\nWHO: Technical and Operational Considerations for Implementing HIV Viral Load Testing. 2014. Reference Source\n\nWHO: WHO/HIV RESNET HIV drug resistance laboratory strategy. 2010. Reference Source\n\nWHO: Hiv drug resistance report 2019. 2019. Reference Source\n\nZhou Z, Wagar N, DeVos JR, et al.: Optimization of a low cost and broadly sensitive genotyping assay for HIV-1 drug resistance surveillance and monitoring in resource-limited settings. PLoS One. 2011; 6(11): e28184. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "71023", "date": "14 Sep 2020", "name": "Graeme Brendon Jacobs", "expertise": [ "Reviewer Expertise Virology", "HIV", "Infectious Diseases" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study analysed the costs involved for HIVDR testing in Nairobi, Kenya. Such studies are vital to maximize the use of available resources for laboratory assays, and to help optimize the success of HIV antiretroviral therapy outcomes.\n\nAIDs – S should be capital letter - AIDS.\n\nIt should be noted that adherence is also a threat to the success of ART, along with the development of resistance.\n\nCD4 cell counts are not recommended anymore – test-and-treat campaigns.\n\nSay that DTG is an Integrase Inhibitor.\n\nThe study gives an overall adequate outline of the major costs involved for HIVDR. As newer technologies, such as Next-generation sequencing, become more freely available (and cheaper), cost analyses studies can be re-evaluated.\n\nIs the work 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": "71361", "date": "16 Sep 2020", "name": "Joses Muthuri kirigia", "expertise": [ "Reviewer Expertise Economic evaluation", "cost-effectiveness analysis", "cost-utility analysis", "cost-benefit analysis", "hospital and programme costing", "health financing", "health economics", "health systems performance assessment", "national health research systems" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nRecommendation to the Editor: This is an important and pertinent manuscript in the ongoing fight against HIV/AIDS. The estimated unit cost is important for planning and budgeting purposes. Therefore, I recommend acceptance after some minor suggested revisions.\n\nResearch Article\nIs the work clearly and accurately presented and does it cite the current literature? Yes.\n\nIs the study design appropriate and does the work have academic merit? Yes. It is a cost analysis of HIV drug resistance tests study in Kenya.\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes. However, I suggest that the authors explain more about the method used to estimate the annual cost of capital inputs. Please see the detailed suggestion to the authors below.\n\nIf applicable, is the statistical analysis and its interpretation appropriate? The statistical analysis is not applicable for this kind of study.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes, the study references the repository (figshare) where data has been deposited.\n\nAre the conclusions drawn adequately supported by the results? Yes, the conclusions are adequately supported by the results.\nOther discretionary suggestions for the authors\nAbstract\nUnder Results section (page 1), amend the last sentence slightly to read as: “A user-initiated modification of halving reagent volumes for some laboratory processes (amplification and sequencing) reduced the unit cost for a HIVDR test to $233.81, i.e. a 13.97% reduction.”\n\nMAIN TEXT\nBACKGROUND\nNo amendment suggested.\n\nMETHODS Data collection and analysis\nOn page 3, first paragraph, the third sentence reads “Cost data were collected on annualized depreciation for capital items including laboratory equipment and furniture, long term training and information technology equipment at a depreciation rate of 10%, …”.\n\nSuggestions (A): I suggest that the authors explain more about the method used to estimate the annual cost of capital inputs. As Drummond et al. explain, capital inputs are assets that are used over periods of more than one year; and depreciate (wear out) with time. Capital costs have two components. (1) Opportunity cost: lost opportunity to invest money used to purchase a capital input to yield positive benefit, e.g. the money could have been put in a fixed deposit to earn interest income. (2). Capital input depreciates over time – thus, its value decreases the longer the life span. Thus, costing of capital inputs should capture both components. As Drummond et al explain, there are two best methods costing capital inputs: (1) To calculate the equivalent annual cost, i.e. annuitize the initial cost of purchase of equipment (or building) over its useful life (years). In calculating the equivalent annual cost, one should take into account both the capital input useful life (in years) and a discount rate. (2) Where competitive market exists to use the rent (per square metre) for building space, and lease price for equipment. The rental and lease price would capture both the depreciation and the opportunity cost. The accounting approach of dividing the price of a capital input by the length of its useful life does not capture fully the two cost components mentioned above. Reference: Drummond MF, Sculpher MJ, Torrance GW, O’Brien BJ, Stoddard GL. Methods for the economic evaluation of health care programmes (3rd Edition). Oxford: Oxford University Press; 2005.1 In Drummond et al., refer to pages 64 and 65 for explanation and pages 72-75 for the formula for estimating the equivalent annual cost of a capital item.\n\nSuggestion (B): Authors should justify the choice discount rate of 10%. The authors cite a published article that has used the same discount rate, and that would suffice.\n\nSuggestion (C): On page 3, first paragraph, the fourth sentence reads “Cost details were obtained from quotations, invoices and delivery notes.” Please replace the word “Cost” at the beginning of the sentence to “Price”.\n\nData analysis\nPage 4: Please consider modifying the first sentence to read as follows: “We developed a costing model in Excel Software (Microsoft, New York) to aid in the estimation of the unit cost and cost for the various categories and laboratory processes.”\n\nSensitivity analysis\nPage 4: The first sentence in this subsection reads “One-way sensitivity analysis for 20% variations to cost categories was performed…”. Authors could cite published studies to justify the choice of 20% variation.\n\nRESULTS\nSuggestion (A): Page 4: I suggest that the authors consider moving subsection titled “HIVDR testing process” and the first sentence of subsection titled “HIVDR unit cost” to the methods section.\n\nSuggestion (B): Slightly amend the Title of Table 1 to read as: “Table 1. Cost breakdown for each category (US$)” Please reflect the minor amendment in Tables 2, 3, and 4.\n\nSuggestion (C): The Figures 1, 2, 3, and 4 portray results that are already contained in Tables 1, 2, 3, and 4. Therefore, I suggest for authors to choose between presenting results either Tables or Figures.\nCost of the modified HIVDR assay\nSuggestion (A): Page 5: Please move the first sentence to methods section.\n\nSuggestion (B): Page 5: Insert the percentage change of “13.97%” into the second sentence. Such that it reads as follows: “The unit cost as a result of halving reagents volumes at the amplification and sequencing step was $233.81, a 13.97% reduction from $271.78 of the original assay.”\n\nChallenges and lessons learnt\nIn Table 5, rephrase the fifth challenge to read as “Frequent electric power outage”. Rephrase the second lesson learnt to read as “Strategic memorandum of understanding between the laboratories and the suppliers”. Rephrase the third lesson learnt to read as “Underutilised equipment can be used for other programmes”. .\nSensitivity analysis\nSuggestion: Page 5: In the sensitivity analysis subsection, please delete the first two sentences because they were already stated in Methods.\n\nDISCUSSION\nPage 5: The ninth sentence reads as “Considering the reagent and consumable costs, there was a correlation in the cost results for these items, as our study estimated the cost to be $101.50.”\n\nSuggestion (A): Please reword the sentence to remove the word “correlation”. I guess you want to say that your finding is very similar to those from cited studies. Correlation is a statistical term.\n\nSuggestion (B): Page 6: Please move the first sentence of the second paragraph to the Methods section. The sentence I am referring to starts as “To answer the question of the cost drivers for HIVDR testing..”. Page 9: In the second paragraph, the second and third sentences read as “On the cost of the HIVDR testing, there was a significant reduction in the cost from $271.78 to $247.30; a ~13.97% reduction in the cost per test. This assay modification led to a ~37.68% reduction in reagent costs.”.\n\nSuggestion (C): Page 9: In the Results section, I could not find the result of $247.30. The percentage change from $271.78 to $247.30 is not ~13.97% but 9.1%. Also, I wonder whether the use of the symbol “~” is appropriate. Please check.\n\nSuggestion (D): Page 9: Should the third sentence read as follows “Unlike HIV viral load and EID, HIVDR testing is not included in the Global Access Program, which could have helped in the scaling up of HIV viral load and EID testing in Kenya at a relatively low cost (WHO, 2014).” Incase, the sentence is correct as it was, please ignore this suggestion.\n\nCONCLUSION\nPage 9: Please consider modifying the first and second sentences as follows: “The MIDRL has implemented HIVDR testing capacity for patients with resistance to ART at a cost of $271.78 per test. The most important cost driver is expenditure on capital inputs, which is likely to reduce when utilization of the equipment increases.”\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-793
https://f1000research.com/articles/9-782/v1	28 Jul 20	{ "type": "Research Article", "title": "Precision medicine implementation and research-practice partnerships: implications of measurement scale differential item functioning", "authors": [ "John J. O. Mogaka", "Moses J. Chimbari", "Moses J. Chimbari" ], "abstract": "Background: Omics-based biomarkers (OBMs) inform precision medicine (PM). As omics-based technologies gradually move into clinical settings, however, a co-occurrence of biomedical research and clinical practice is likely an important variable in the implementation of PM. Currently, little is known about the implications of such research-practice co-occurrence. Methods: This study used data collected from a pilot study designed to inform a full-scale PM implementation study through the validation of the measurement tool. It applied item response theory (IRT) methods to assess the tool’s reliability and measurement invariance across two study subgroups associated with research and practice settings. Results: The study sample consisted of 31 participants. Measurement invariance assessment was through differential item functioning (DIF) analysis with bootstrapping through Monte Carlo simulation. Overall, 13 out of 22 items that formed the PMI scale had DIF at significance level α=0.25. Item response functions (IRFs) revealed how each subgroup members responded to scale items and their attitudes towards factors that influence PM implementation. Conclusions: Attitudinal similarities and differences towards factors influencing PM implementation amongst those in biomedical research as compared with those in practice were established. Results indicated PM implementation knowledge that is unique and common to both groups. The study established the validity and reliability of the new PM implementation measurement tool for the two subgroups.", "keywords": [ "precision medicine implementation", "omics technologies", "implementation measurement", "differential item functioning", "item response theory" ], "content": "Introduction\n\nRecent advances in omics technologies offer greater understandings of how gene-environment interactions affect health and pathological processes. Omics-based biomarkers (OBMs) resulting from advances in biomedical technologies form the basis of precision medicine (PM), an approach to medicine that emphasizes predictive, preventive and personalized medical care1. Deep insights gained in systems biology, molecular evolution and microbial environments is increasingly being applied in clinical settings through OBMs, with huge biomedical, clinical and public health implications. For instance, due to broadening understanding of the complex genotype-lifestyle-environment interactions, how biomedical research evidence is collected and applied is changing to reflect a shift from “average many” to “unique few” approach to health interventions. Evidence-based medicine (EBM)2, the widely applied and accepted approach to medicine, is mostly informed by systematic reviews, meta-analyses or group-level studies from which average recommendations for the “average person” are derived. However, this “one-size-fits-all” approach has severe limitations in that it does not adequately cater for uniqueness in individuals, neither does it consider disease heterogeneity in larger populations. Although most people may be accounted for within some average categories, yet many others are ‘outliers’, falling outside recommended mean estimates. Although understanding individual-level underpinnings of health and disease at finer resolution may be overwhelming in practice, PM provides a shift away from the generalized average protocols to personalized strategies in medical care. Moreover, PM, through OBMs and other biotechnologies, enables better disease delineation and patient stratification. In emphasizing individual uniqueness at the molecular level and tailored care based on uniqueness of genotype, PM also enables advanced detection of pre-symptomatic conditions in the population and individuals, while improving surveillance and management of infectious diseases. This results in better-adapted and personalized therapies, significantly delayed disease onset, as well as disease prevention.\n\nHowever, while interventions associated with PM, including cell-, gene-, and tissue-engineered therapies3 are highly personalized in nature, their clinical validity and utility is generally limited. This lack of adequate clinical utility implies that in PM, the boundary between research and practice is blurred as opposed to the traditionally more pronounced research-practice boundary in EBM. Despite mounting evidence supporting the value of OBMs in improving health outcomes (e.g., Berg AO4), the moving-target nature of most genomic-based interventions implies that the evidence base for PM must constantly be updated as more knowledge is gained over time. Implementation of PM therefore inevitably juxtaposes biomedical research and clinical practice elements, implying co-existence of both research and practice settings. Although such co-occurrence settings create novel opportunities to advance medicine through PM, little is currently known about the nature and implications of such research-practice partnerships, especially for PM implementation at health systems level.\n\nWe assessed a PM implementation (PMI) measurement model using a latent variable approach informed by item response theory (IRT). The study constructed four variables (factors) that were hypothesized to have varying influences on PM implementation at health systems level. These were: 1) Characteristics of omics biomarkers; 2) Organizational support in terms of resources required for implementation; 3) Public genomic awareness; and 4) precision medicine implementation outcomes. OBMs are biological disease- or health- status indicators gleaned from genomics (gene structure and sequencing), epigenomics (gene expression via transcriptomics)5, proteomics (RNA products)6,7, metabolomics ( metabolites) and DNA-based tools that detect bacteria, parasites and viruses that coexist in the human body through metagenomics8. Despite apparent advantages associated with omics biomarkers, varying perceptions about their effectiveness persist, largely due to less-conclusive clinical validity. Concerns around OBMs include questions around the biomarkers’ reliability, interpretation of test results and quality standards9. Based on existing literature, six items were selected as indicators for the ‘Omics Biomarkers’(OBM) construct as illustrated in Table 1. Organizational support relates to resource investments required to move OBMs from point of discovery to point of clinical application. For instance, material and non-material investments are needed to continuously update existing biomedical knowledge including new algorithms and bioinformatics methodologies, as well as high-velocity and high-capacity computation facilities. Resources are also required in compliance with good practice in genomic data governance through optimized data capturing, storage, transfer and processing. Organizational support, therefore, is a factor that can influence PM implementation outcomes. Six items were selected as its indicators (Table 1). On the other hand, although use of biomarkers is beneficial in circumstances such as identifying susceptibility for inherited conditions, this often implies genetic profiling, which can lead to anticipation, skepticism and concern at the personal level10,11. Individuals, empowered through publicly available information prevalent in social media and other connected technologies, may advocate for adoption of certain biomedical technologies. They may also manifest willingness to participate in genomic studies, as they appreciate relative importance of doing so not only for themselves but for public good in general. Therefore, the level and quality of public genomic awareness give rise to differing genomic attitudes, perceptions and values among members of the public, both as individuals and as health providers. Six items were selected to be indicative of ‘Public Genomic Awareness’, as seen in Table 1. Finally, the PM implementation outcomes factor captures the long-term goal of PM implementation efforts: improved uptake and routine use of OBMs in healthcare. Four items were selected as measures of the construct ‘Implementation Outcomes’ (Table 1).\n\nAs omics-based technologies gradually move into clinical settings, a co-occurrence of biomedical research and clinical practice is likely to present as an important variable in the implementation of PM. This co-occurrence, however, is complicated by potential differential interpretation of factors that affect or influence PM implementation. Modern psychometric tools offer reliable means of assessing item-level measurement variability that focus on differential item functioning (DIF)12. DIF has been defined as a measure of different probabilities of success or endorsement across construct while controlling for the underlying trait being measured13. DIF represents unequal response patterns among groups, which can profoundly threaten research findings, as it may constitute bias. To assess this kind of differential item functioning in PM, this paper sought to establish a PM implementation measurement tool’s differential item functioning (DIF) across two study participant sub-groups; one affiliated to academia (representing research) and the other affiliated to industry (representing practice). In as much as measurement variability due to sub-group membership - irrespective of the construct being measured - has often been underscored in other disciplines14, it is important to assess the same in the new field of PM. Item-level invariance assessment entails a valuable exercise as it serves to test what may often be implicit assumptions concerning much measurement processes15. Furthermore, such assessment will help curtail a number of problems including errors in hypothesis testing, flawed population forecasts and policy planning16 in relation to PM implementation. The study also confirmed the tool’s psychometric reliability and construct validity.\n\nTable 1 shows the four PM implementation factors (latent variables) and items that measure them.\n\n\nMethods\n\nThis was a cross-sectional study that employed analytical approaches for quantifying factorial relationships. DIF analysis helped to reveal differences in perceptions towards PM implementation between two study participant subgroups: one affiliated to academia and the other affiliated to industry. Item response theory (IRT) is one of the widely used methods for assessing DIF. In this study, IRT allowed for a critical examination of the information that was harvested from the manner study participants endorsed and ranked the various items associated with PM implementation. As noted earlier, DIF examines the probability of endorsing an item conditioned on the latent trait. This method was deemed suitable for examining DIF in this study as IRT examines the monotonic relationship between responses and the latent trait17. Furthermore, as compared to classical test theory, IRT parameter estimates are not only as confounded by sample characteristics, but statistical properties of items can be expressed with greater precision, thereby increasing the interpretation accuracy of DIF between sub-groups18.\n\nMembers of “academia” were defined as individuals involved in biomedical research that related to translating newly discovered molecular or omics-based biomarkers (OBMs) for purposes of clinical or population health use. Members of “industry” were defined as individuals involved in the clinical use of the biomarkers or those involved in commercialization activities related to OBMs (e.g., Direct-To-Consumers Genetic Testing, DTC-GT)19. “Precision medicine implementation” was defined as the process of translating newly discovered omics-based biomarkers (OBMs) for clinical or population health use. “OBMs” were defined as candidate genetic biomarkers that are in the process of being clinically validated or those already validated and in clinical use.\n\nThe snowball sampling method, a non-probability sampling method, was applied in identifying potential study participants from population of interest. Inclusion was based on a participant’s affiliation to either an academic institutions or a commercial entity (industry); the institution or organization had to be involved in molecular/genetic testing and/or omics-based biomarkers in Africa; a participant had to be involved in the field of precision medicine and/or biomarker-related biomedical research as indicated by contribution to the field through publications in peer-reviewed journals and/or attendance of related academic conferences. It is well known that snowball sampling poses significant recruitment “community bias” risk whereby recruitment of new members does not branch out from the sample subgroup of individuals initially accessed. In the present study, this risk of source bias was addressed by properly selecting the initial individuals to ensure proportional inclusivity of subgroups in the seed (initial) sample. Consequently, the seed sample was identified and selected from a precision medicine-related conference; an event we regarded as representative of our study population as it brought together diverse groups from academia and industry involved in precision medicine and its implementation in resource-constrained settings. Therefore, the probability of selection bias for subsequent in-link recruitment waves of study participants was negligible. Besides, it has been shown that with an appropriate bootstrap resampling, selection bias due to the initial seed sample (if known to be representative of the targeted population) is progressively attenuated as the sample expands wave by wave20,21.\n\nGuided by general principles of the Nuremberg Code, the Declaration of Helsinki and institutional review board permit obtained from the University of KwaZulu-Natal (BREC Permit Ref No BE513/18), a study package was distributed via email to potential participants between June and July 2019. Since this study contained negligible risk of potential embarrassment or other ethical dilemmas that are usually associated with snowball sampling in many other studies, initial participants were encouraged to forward the email containing the study package to their colleagues. The study package (available as Extended data)22 included an invitation letter with study description, consent form and a link to the online card-sort platform. A total of 31 study participants were recruited for the study. Although there is no definitive number for a pilot study sample size according to Hunt, Sparkman and Wilcox23, to increase statistical power and degrees of freedom for our study, we applied a Monte Carlo simulation (MCS) approach as described by Mooney24. MCS studies are computer-driven experimental investigations in which certain parameters, such as population means and standard deviations that are known a priori, are used to generate random (but plausible) sample data24. This method of generating and analyzing data treats the collected research sample as a “population reservoir” from which a large number of random samples are drawn with continuous replacement such that the probability of selection for any given case remains equal over every random draw25. We requested 2,000 iterations, drawn with replacement from the original data set of 31 cases (our empirical sample).\n\nA card-sort task approach26 was used to obtain comparative item-rating data. Respondents were presented with a set of cards that contained constructs clearly defined using everyday language and a set of categories onto which the concepts on the cards were to be mapped, as explained by Angleitner, John, & Lohr27. The respondents were asked to read each item and assign it to the construct or concept it best indicated, in their judgment. Participants were required to rank the cards according to their own understanding and preference, indicating their perception on concepts presented on each card. If participants deemed it necessary to make any changes as to where a card previously assigned should be reassigned to, they could do so. Although simple in practice, this exercise generated enough data that was used to assess the desired group perception levels, construct validity and scale reliability. The actual sorting of the items was, however, performed using an online platform hosted and supported by Optimal Workshop28. The items are presented in Table 1. Besides offering convenience to study participants, the online platform enhanced data security and confidentiality. A total of 22 items were to be assigned into four categories initially provided for, with participants allowed to create own separate construct categories if they so desired. The results about number of items per category and the items’ ranking order (positioning) within their respective categories were then extracted and analyzed. Participants’ demographic data were also extracted, including sex, highest educational qualifications and age. Data was subsequently subjected to item response theory (IRT) analysis29,30 with logistic regression modelling.\n\nIn summary, the data are an output of an exercise where respondents were required to place items arranged as cards into specific categories and ranked in order of perceived importance. The cards related to specific attributes indicative of wider factors thought to influence precision medicine implementation at health systems level. This meant that if an item on the card was regarded as the most important in each category, it was ranked 1st, and the next ranked 2nd and so on. If a card was not placed in a category, it was scored zero in that same category. For compactness and statistical purposes, the data was formatted to have one observation per row (for 31 participants, there were 31*4 = 124 observations).\n\nOne of the assumptions in item response theory (IRT) model analysis is that factors are generally unidimensional31. Therefore, to examine differences in perception between the two sub-groups (academic and industry) related to each item in the PM implementation scale, DIF was tested within each subscale. Each of the four factors that were identified as influencing PM implementation correspond to a subscale. Thus, four sets of tests were conducted with each of the four factors (sub-scales) examined separately for both item response functions (IRFs) and unidimensionality. To determine and confirm sub-scale unidimensionality, we used R package “mokken” version 2.8.1132. In this study, scale reliability referred to the consistency of measurement, while construct validity refers to the extent to which a set of indicators that were devised to gauge the four constructs (OBM, OrG, PGA and ImO) really measured that construct. Differential item functioning (DIF) was defined as a property of an item that shows the extent to which the item might be measuring different dimensions of the variable it was supposed to measure for members of separate subgroups. We applied the ordinal logistic regression modelling function in the package “lordif” (logistic ordinal regression differential item function) using IRT, version 0.3-3)33 in R version 3.6.134. The package “lordif” provides a logistic regression and IRT framework for detecting various types of differential item functioning33. We preferred it because it could adequately handle the item-sort-and-rank data that we used. To analyze DIF in the scale, we devised three models. The first of the three models distinguish DIF and non-DIF items. The second model identifies uniform-DIF items that occur due to an item tapping a dimension of the attribute measured in the scale that manifests differently between the groups. That is, for uniform DIF, whereas the probability of endorsing an item at a certain ranking is greater for one group than the other, this is uniform over all levels of knowledge of the factors under consideration. Finally, the third model identifies non-uniform DIF; that is the probability of endorsing an item at a certain ranking is greater for one group than the other, but this is not uniform over all levels of knowledge of the factors under consideration. This may be because of distracting elements (not necessarily related to the scale) in the measurement process, such as different understandings of a word or phrase used on the item35.\n\nThese three models can be summarized as36:\n\nlogit(P(U = 1)) = β0 + β1θ + β2g + β3θg (Model 3: Non-uniform DIF)\n\nlogit(P(U = 1)) = β0 + β1θ + β2g (Model 2: Uniform DIF)\n\nlogit(P(U = 1)) = β0 + β1θ (Model 1: No DIF)\n\nwhere:\n\n(P(U = 1)) is the probability of endorsing an item at a particular rank in a category,\n\nθ is the participant’s latent perception level on the factor the item is measuring,\n\ng is the group to which the participant belongs (academia or industry).\n\nWe used two sets of logistic regression-based criteria for detecting DIF: one set based on statistical significance (likelihood ratio χ2 tests) and the other on Pseudo R2 magnitude (Nagelkerke's and McFadden’s pseudo R2). Uniform DIF was examined by comparing the log likelihood of model 1 with 2 (degree of freedom (df)=1) and non-uniform DIF by comparing model 2 with 3 (df=1).The comparison between model 1 and model 3 (df=2) detected the total DIF effect—both uniform and non-uniform DIF36,37. Items that revealed significance in any of the three likelihood ratio χ2 tests with an alpha of 0.25 (Model 1 versus 2, Model 2 versus 3, and Model 1 versus 3) were flagged as having DIF. To examine the magnitude of DIF, we used various pseudo R238,39 such as Nagelkerke's Pseudo R2 and McFadden’s pseudo R2 statistics for the defined model comparisons, and applied Zumbo et al.’s guideline to evaluate R240: effect size was regarded as negligible DIF (pseudo R2 < 0.035), moderate DIF (0.035 ≥ pseudo R2 < 0.070), and large DIF (pseudo-R2 ≥ 0.070).\n\nFor individual participants, the difference between the DIF-adjusted (purified) subscale score and the initial unadjusted score for each subscale. Finally, we obtained item and subscale characteristic curves to show the impact of DIF for each group, i.e. item parameter estimates were computed and graphically examined via item characteristic curves (ICCs) and other graphs. R code used to calculate DIF is available as Extended data41.\n\nDescriptive statistics that were obtained for the data included analysis of outliers, which was defined as any value that is greater than three standard deviations above or below the mean. Multivariate skewness and kurtosis statistics of the data were assessed using R package ‘psych’ version 1.8.1242.\n\n\nResults\n\nSkew values ranged from 1.21 to 2.41 while kurtosis (k) statistics for 21 items ranged from -0.37 to 2.49, with the 22nd item indicated at k= 5.87. Figure 1 is a quantile-quantile (Q-Q) plot formed by the data and shows the distribution of the data against the expected normal distribution. Since the observations seemed to approximate a straight diagonal line with minimal deviations, we concluded that our data has a general normal distribution. Despite one item being an outlier with k = 5.87, its inclusion in the data set did not alter results, and therefore all items were included for further analyses.\n\nThe data did not contain any missing values because all the fields of the online item sort tool for data collection were set as “required” to eliminate non-responses. Underlying data are available at Zenodo22.\n\nOf the 35 participants invited and who attempted the item-sort exercise, 31 successfully completed it (88% response rate). There were three more female participants than males (10%). Those with research qualifications (PhDs and equivalent) were the majority (55%), while slightly a quarter of the participants were medical doctor. The rest of participants’ qualities varied greatly and are summarized in Figure 2.\n\nThe correlogram in Figure 3 is an inter-item correlation plot indicating item and scale structure in the data. In the upper triangle, positive correlations are displayed in blue and negative correlations in red colored circles. Color intensity and the size of the circle are proportional to the correlation coefficients, helping to identify “groups” of variables that share a strong relationship with each other (hierarchical clustering). The lower triangular correlation matrix displays the actual correlation values.\n\nEach item seems to fit into one of the four well defined clusters (Figure 3), implying four factors defined the data as hypothesized. Items x16 (“Using this genetic/omics test has been regarded by practitioners as an appropriate mechanism for patient management (e.g. aid in drug dosage decisions, in carrying fetuses to term or carry out prophylactic surgery)”; and x20 (“Practitioners are more willing to order the genetic/omics test more often whenever deemed necessary”) are the only items not showing a strong correlation within the groups they belong.\n\nTo confirm the suggested four factors in the variable correlation matrix, we performed further scale analysis using functions in the “mokken”43 R package. Four unidimensional scales were identified as dimensionality solutions via the package’s automated item selection algorithm (AISP) performed at factor loading ≥0.3 and Type I error level (α)=0.05. Table 2 shows the results of this procedure and various reliability associated with each factor (subscale).\n\nKey: MS=Molenaar Sijtsma statistic (a.k.a. rho); Alpha=Cronbach's alpha; Lambda.2=Guttman's lambda.2; OBM=Characteristics of omics biomarkers construct; OrG=Organizational support construct; PGA= Public genomic awareness construct; ImO = PM implementation outcome construct. ‘Chi12’, ‘Chi13’, and ‘Chi23’ denote the likelihood ratio χ2 statistic between Models 1 and 2, Models 1 and 3, and Models 2 and 3, respectively; R12, R13, R23 indicate Nagelkerke's pseudo R2 from comparing Models 1 and 2, Models 1 and 3, and Models 2 and 3, respectively. Scale dimensionality solution was obtained by the automated item selection algorithm (aisp).\n\nSub-scales were demarcated by means of scalability coefficients44 as indicated by the coefficient H in Table 2. At the lowest scalability threshold of 0.3, four unidimensional subscales were identified through the AISP. AISP is a scaling method of item selection we applied to partition the 22 items into subscales. This implies that there were four latent factors (subscales) measured by the 22 items (i.e., four underlying variables explain the association between latent factors and responses to items). Unidimensionality ranges between H=0 and H=1: strong unidimensional scale has H ≥ 0.5, moderate 0.4 ≤ H < 0.5, and weak 0.3 ≤ H < 0.444. The four subscales’ coefficients H ranged between H ≥ 0.5 and H = 0.8, implying the subscales were strongly unidimensional. This was strongly supported by the reliability indices (Mokken’s rho, alpha and Guttman’s lambda-2 (λ2)) which indicated high internal consistency (internal validity/goodness-of-fit) for the four subscales. Cronbach's α, the expected correlation of two tests that measure the same construct, was separately obtained for the four constructs. All four subscales had coefficient alpha (α) ranging between 0.76 and 0.95 (with an average α =0.88). Besides, given that internal scale consistency (intercorrelations among items) is maximized when all items in a subscale measure the same construct, we concluded that the high Cronbach's alpha values for our subscales are an indirect but confirmatory indicator of the degree to which the items in the subscales measure corresponding unidimensional latent construct. However, Cronbach's alpha has been found to have limitations in measuring reliability in some instances45–47. Due to this concern, we obtained other reliability indices for the same purpose as described below.\n\nGuttman’s λ2 is a reliability estimate that is more robust than Cronbach’s alpha, i.e., more resistant to outliers. The range of λ2 values for the four subscales were: 0.81 ≤ λ2 ≥ 0.96. This implies that more than 80% of variance in each of the subscales is attributable to the true score and less than 20% to error, confirming that the internal construct validity and scale reliability is good. On the other hand, Mokken’s rho, considered to be an improvement in relation to Cronbach’s alpha and whose values should exceed 0.70 to show adequate scale reliability48, was obtained. As shown in Table 2, all the four subscales had their rho (MS) coefficients above 0.78, confirming good scale reliability.\n\nTable 2 also shows DIF detection outcomes using logistic regression approach33. DIF items revealed from the likelihood ratio χ2 tests are indicated with asterisks in Table 2. Of the 22 items making up the PM implementation (PMI) scale, 13 were flagged as DIF items at α=0.25 with MCS set at 2,000. They all showed statistical significance for total DIF effect test when comparing Models 1 and 3. Of the 13 DIF items however, seven were indicated as mixed DIF items. Their likelihood ratio χ2 test comparisons between Model 1 and 2, and models 2 and 3 were statistically significant. Moreover, of the remaining six, five had non-uniform DIF (x1, x4, x9, x16 and x20); the comparisons were statistically significant for Models 2 vs 3 as compared to Models 1 vs 2. Only one item had uniform DIF (x19): it showed non-significant comparisons for models 2 vs 3 but significant comparisons for models 1 vs 2. However, using the same statistical significance test, all items were DIF flagged at α =0.5 and non at α =0.01. This finding indicates the effect of statistical significance levels in DIF detection.\n\nHowever, in contrast to the likelihood ratio χ2 tests that detected 13 of the 22 items as DIF-positive, Nagelkerke's Pseudo R2 statistics flagged only five items as DIF (x1, x4, x7, x10 and x12). These were all moderate DIF (0.035 ≥ pseudo R2 < 0.070). To verify this result, we inspected other pseudo R2 statistics, such as McFadden’s and Cox and Snell’s R2. The additional pseudo R2 indices also yielded similar values.\n\nFrom the above analyses, we generated various graphs. However, due to space constraints, we present only those graphs related to characteristics of omics biomarkers (OBM construct). Diagnostic plots are used below to describe the relationship between the perception latent trait and the probability of responding positively to an item as observed from the data (positively here means endorsing an item in any of the four scales). Participant’s perception trait level is signified by theta (θ).\n\nThe top left plot in Figure 4a shows item true score functions based on group-specific item parameter estimates. It shows the item characteristic curves (ICCs) for the item for industry vs academia affiliated participants represented with red dashed and black solid lines respectively. The slope of the function for industry group was substantially higher than that for academia group, indicating non-uniform DIF. The upper–right graph shows the absolute difference between the ICCs for the two groups, indicating that the difference is mainly at high levels of perception of omics biomarkers (especially between average (0) ≥ θ ≤ +3). The lower-left graph shows the item response functions for the two groups based on the affiliation–specific item parameter estimates (slope and category threshold values by group, as also printed on the graph). It juxtaposes the item response functions for academia and industry groups. The non-uniform component of DIF revealed by the LR χ2 test can also be observed in the difference of the slope parameter estimates (33.46 vs. 2.75). The lower-right graph shows the absolute difference between the ICCs (the upper-right graph) weighted by the score distribution (size) for the focal group, i.e., industry group, indicating minimal impact.\n\n(a) Graphical display of the item “X1:The omics test has been used among people similar to the present target population” which shows non–uniform DIF with respect to affiliation to either academia or industry. (b) Box-and-whisker plot and a scatter plot showing (individual-level) differences between initial and purified scale scores for each respondent. (c) Impact of DIF items on test characteristic curves (TCC) for all items and DIF items. (y-axis = sum of the item scores of the OBM domain; x axis (theta) = knowledge of OBMs). (d) Smoothed histogram showing perception differences towards omics biomarkers between those affiliated to academia and industry.\n\nFigure 4b displays the boxplot and a scatter plot that show DIF items as unique to each group, accounting for DIF in trait estimates. It shows the difference in latent trait level between the initial IRT-based trait estimate with ignored DIF items and the purified trait estimate with DIF items accounted for. Each (black) circle and (red) triangle stand for the latent trait estimate of the OBM domain for a single participant. It also shows the expected amount of change in latent trait estimates when DIF is accounted for. Accounting for DIF items in a multi-item scale is commonly referred to as “scale purification”. In both graphs, the y-axis is the difference (initial – purified) and the x-axis on the right graph is the initial latent trait level. The Box-and-Whisker plot on the left shows that median difference was around -0.04, and the middle 50% of respondents, depicted as the box (i.e., the interquartile range)ranged from approximately -0.04 to 0.07, indicating a fairly normal but slight right-skewed distribution with possible outliers at high ends (θ above average). The scatter plot on the right shows the initial (no DIF) vs purified (DIF accounted for) difference against the initial latent trait level that ignored DIF, implying that across the entire latent trait continuum, those participants who were affiliated to industry (red triangles) show more positive difference, further suggesting that accounting for DIF leads to lower scores for them compared to initial (non DIF) scores; for those affiliated to academia, (dark circles), the pattern is in the opposite. Guidelines are placed at 0.0 (solid line), i.e. no difference, and the dotted horizontal reference line is drawn at the mean difference between the initial and purified estimates.\n\nWhat is suggested in Figure 4b corresponds well with Figure 4c, the test characteristic curves (TCC). The y-axis of Figure 4c indicates all possible scores for domain “OBM”. Figure 4c indicates a clear difference in TCC curves, albeit in small margins, of DIF item responses between academia and industry groups. On the other hand, the left graph for all items displayed a much smaller difference than the right graph of Figure 4c. This can be explained as the DIF impact being diluted by the non-DIF items, resulting in the overall score difference between the two groups becoming minimal. It is evident that at average knowledge levels (θ = 0), the scale performs better among those affiliated to industry than academia. But at higher trait levels (θ ≤ +2), industry lags behind academia.\n\nFinally, Figure 4d shows smoothed histograms of the omics biomarkers perception (knowledge) levels for those study participants affiliated to industry (dashed red line) and academia (solid black line) as measured by the OBM subscale (theta). Industry affiliated participants on average had lower mean scores than their academia-affiliated counterparts, although there is a broad overlap in the distributions.\n\n\nDiscussion\n\nItems related to public genomic awareness (PGA) appear to have the least DIF. In contrast, all the factors (characteristics of omics biomarkers (OBM), organizational support (OrG) and precision medicine implementation outcomes (ImO)) had a substantial number of items flagged for DIF. There were two types of DIF items that identified in this study: uniform and non-uniform. The only item shown to have uniform DIF on the scale was item x19 (“The genetic/omics test is yet to be used as a routine practice within its intended service setting”); implying that one subgroup was consistently more likely than another to endorse this item at each level of the measured trait. Five items that showed explicit non-uniform DIF implied that there was inconsistent pattern of response; i.e., one group crossing-over, so that at certain levels of the trait, one group was more likely to endorse the item, while at another level, the other group was more likely to endorse the same item. Public genomic awareness is closely related to social and ethical nuances of research in precision medicine49,50. PGA focuses on ethical questions ranging widely: from social justice issues regarding healthcare equity and access to specific questions regarding consent, safety and public acceptability of genomic technologies. The level of omics technology acceptability among practitioners (physicians) is another aspect of PGA51. Our study findings agree with other findings in literature that focus on genomic engagement with public and practitioners49,50,52,53. The PGA non-DIF items indicate deep-cutting agreement across study participants on the likelihood of inadequate genomic knowledge and genetics personnel to deal with issues surrounding ethical, legal and social implications of advancements in precision medicine. The fact that both subgroups (academia and industry) mostly agreed on most PGA aspects suggests that in a research-practice partnership designed to advance PM, both groups are likely to have similar outlooks towards ethics and genomics such as the balance between health gains and possible loss of privacy which may warrant sensitive, ongoing attention. The non-DIF items about PGA among study participants also suggests overwhelming support for community consultation and involvement where clinical implementation of omics technologies is concerned.\n\nAssessment of invariance of chosen measurement models across subgroups is likely to emerge central to policy efforts that assess optimal ways of implementing PM at national health system level. This study has helped show the critical importance of a measurement tools’ psychometric evaluation and how DIF analysis can play a vital role in implementation decisions. For instance, depending on the statistical procedure used for DIF detection, differing results may be yielded. Thus, one procedure may indicate DIF for certain items while others do not. The present study’s findings, however, have another policy implication: in as much as evidence of DIF may play a vital role, it should by no means be the sole basis for policy decisions and conclusions. Presence of DIF may be an indication of problematic items that need to be revised or omitted and not necessarily an indication of sub-group irrationality. Therefore, DIF analysis can be considered a useful tool for item analysis but is more effective when combined with robust reasoning. Another consideration in the face of DIF in a measurement model is that a sub-group’s domain score should be interpreted in a way that corrects for the measurement bias. This can lead to two policy implications: either to develop subgroup norms or to understand why DIF items occur in the first place and then to later revise and improve DIF items of the measurement model.\n\n\nConclusion\n\nMore generally, precision medicine combines both practice and research components. The relationship between academic research and clinical practice in precision medicine is, however, not easy and straightforward. In as much as public health and clinical practice through PM may benefit from advances in omics technologies and deeper gene-environment insights that flow from rigorous biomedical research, research also benefits from being informed by practice problems and practical knowledge, leading to co-creation of solutions to broader PM issues in both spheres.\n\nThis study assessed latent traits (factors) that relate to precision medicine implementation using a newly formulated implementation measurement scale in order to validate the scale’s general reliability and construct accuracy. The findings demonstrate general conceptual and psychometric measurement equivalence of the scale across two subgroups involved in PM implementation: academia (research) and industry (practice). Differential item functioning (DIF) analysis was used to study the performance of the 22 items of the PMI measurement scale, examining whether the likelihood of item (categorical) endorsement is equal across the two subgroups as matched on the measured trait. Even though there were no DIF flagging at statistical significance of α=0.5, DIF analysis at α=0.25 revealed the presence of some DIF items in the scale. There were two types of DIF items that were pointed out in this study: uniform and non-uniform.\n\nMoreover, the study delved into the assessment of statistical basis of the measurement model as well as providing various indices such as scalability, reliability and unidimensionality. All four subscales showed strong scalability. The a priori subscales were strongly supported by the automated item selection algorithm, AISP, further validating the theory behind the generation of the measurement items.\n\nApart from highlighting DIF items on a newly developed precision medicine implementation (PMI) measurement tool, this paper also pointed out potential and benign differences between perception on PM implementation held by those affiliated to industry (those in clinical and public health practice) and those in biomedical research. This has both implicit and explicit implications in the field of PM implementation, especially at health systems level. First, it highlighted the kind of information separately held by these two subgroups in the field of PM. Secondly, it also highlighted the kind of information held together in common by these important subgroups in the pursuit of ideals of PM. Thirdly, the findings contained in this paper may inform the kind of information that should be availed to them. With this paper, we hope to have contributed to draw attention to those in PM to the fact that biomedical research and PM practice need deliberative attention from both researchers and practitioners to succeed.\n\n\nData availability\n\nZenodo: Extended data for Manuscript: Precision medicine implementation and research-practice partnerships: implications of measurement scale differential item functioning (DIF). http://doi.org/10.5281/zenodo.392527622.\n\nFile ‘DIF Analysis Raw.Extended Data’ (XLSX) contains the redacted survey responses from the card sort exercise and the results of the card sort exercise itself.\n\nZenodo: Extended data for Manuscript: Precision medicine implementation and research-practice partnerships: implications of measurement scale differential item functioning (DIF). http://doi.org/10.5281/zenodo.392527622.\n\nFile ‘DIF Study Package.Extended data’ (DOCX) contains the study package provided to participants.\n\nZenodo: R code for DIF. http://doi.org/10.5281/zenodo.392533641.\n\nThis project contains the R code for this study in R and PDF formats.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nCollins FS, Varmus H: A new initiative on precision medicine. 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Multiple Linear Regression Viewpoints. 2013; 39(2): 17–26. Reference Source\n\nGelin MN, Zumbo BD: Differential item functioning results may change depending on how an item is scored: An illustration with the Center for Epidemiologic Studies Depression Scale. Educational and Psychological Measurement. 2003; 63(1): 65–74. Publisher Full Text\n\nJohn M: R code for DIF [r code]. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3925336\n\nRevelle W: Procedures for psychological, psychometric, and personality research. Acesso em. 2012; 9. Reference Source\n\nVan der Ark LA: New developments in Mokken scale analysis in R. J Stat Softw. 2012; 48(5): 1–27. Publisher Full Text\n\nPalmgren PJ, Brodin U, Nilsson GH, et al.: Investigating psychometric properties and dimensional structure of an educational environment measure (DREEM) using Mokken scale analysis–a pragmatic approach. BMC Med Educ. 2018; 18(1): 235. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchmitt N: Uses and abuses of coefficient alpha. Psychol Assess. 1996; 8(4): 350–353. Publisher Full Text\n\nSijtsma K: On the use, the misuse, and the very limited usefulness of Cronbach’s alpha. Psychometrika. 2009; 74(1): 107–120. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTavakol M, Dennick R: Making sense of Cronbach's alpha. Int J Med Educ. 2011; 2: 53–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSijtsma K, Molenaar IW: Reliability of test scores in nonparametric item response theory. Psychometrika. 1987; 52(1): 79–97. Publisher Full Text\n\nEtchegary H, Green J, Parfrey P, et al.: Community engagement with genetics: public perceptions and expectations about genetics research. Health Expectations. 2015; 18(5): 1413–25. Publisher Full Text\n\nSamuel GN, Farsides B: Genomics England’s implementation of its public engagement strategy: Blurred boundaries between engagement for the United Kingdom’s 100,000 Genomes project and the need for public support. Public Understanding of Science. 2018; 27(3): 352–64. Publisher Full Text\n\nWonkam A, Hurst S: A call for policy action in sub-Saharan Africa to rethink diagnostics for pregnancy affected by sickle cell disease: differential views of medical doctors, parents and adult patients predict value conflicts in Cameroon. OMICS. 2014; 18(7): 472–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTekola-Ayele F, Rotimi CN: Translational Genomics in Low- and Middle-Income Countries: Opportunities and Challenges. Public Health Genomics. 2015; 18(4): 242–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTiffin N: Unique considerations for advancing genomic medicine in African populations. Per Med. 2014; 11(2): 187–96. PubMed Abstract \| Publisher Full Text" }	[ { "id": "74152", "date": "02 Dec 2020", "name": "Gerald Mboowa", "expertise": [ "Reviewer Expertise 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\nThis article provides insightful background on precision medicine implementation and research-practice partnerships, and suggests implications of measurement scale differential item functioning. The authors describe the methods for precision medicine implementation by employing analytical approaches for quantifying factorial relationships as well as presenting graphical analyses of differential item functioning (DIF) effects.\nThe authors clearly discuss factors (characteristics of omics biomarkers (OBM), organizational support (OrG) and precision medicine implementation outcomes (ImO)) for DIF. This research article is scientifically sound.\nMinor comment for the authors: Authors should discuss the limitations of the study design and the future of Omics-based biomarkers (OBMs).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "90224", "date": "03 Aug 2021", "name": "Ken Asada", "expertise": [ "Reviewer Expertise Multi-omic analysis", "Precision medicine", "Machine learning", "enome and epigenomics", "Cancer research." ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAuthors focused on the gap between research and practice settings of OBM and stated on the importance in health sciences, which I agreed with. Additionally, DIF is probably a suitable method to use in their study. However, manuscript is difficult to fully understand. Authors need to reconstruct the structure and amend the manuscript to convey their purpose and study smoothly to readers.\nHere are my comments.\nStudy design and Procedure should explain more clearly. If needed, please consider using a flowchart.\n\nI have a concern about the data size author used for their analyses.\n\nTable 1: Please revise carefully if the statements are widely accepted. For example, is it truly \"easy to obtain\"?\n\nAuthors stated that \"based on existing literatures, six items were selected as indicators for the 'Omics Biomarkers' (OBM) construct as illustrated in Table 1.\" What literatures do authors refer? Please provide and cite as references. Additionally, please use other abbreviations since OBMs has been already used as \"Omics based biomarkers\" in the early paragraph. OBM and OBMs are quite similar terminology and making confusion. On top of that, I see the following sentences (1) six items were selected as indicators for the 'Omics Biomarkers' (OBM) construct as illustrated in Table 1. (2) Six items were selected as its indicators (Table 1). (3) Six items were selected to be indicative of 'Public Genomic Awareness', as seen in Table 1. (4) Four items were selected as measures of the construct 'Implementation Outcomes' (Table 1). It is of your interest but I suggest that authors should make more clear explanation for Table 1.\n\nAbbreviations: Some sentences use abbreviation without explanation before use. On top of that, abbreviations are repeatedly showing up in the manuscript. For example, \"differential item functioning (DIF)\" shows up in page 3, page 4, page 6, and page 12. So does IRT. \"item response theory (IRT)\" has repeatedly appeared, which once in page 5, and twice in page 6. I assume it could be more in other pages. Again, explanation of the abbreviation once is enough.\n\nAuthors focused on the importance and gap between biomedical research and clinical practice. DIF is the one authors claim to fill in the gap between biomedical research and clinical practice. Actually, some papers have been already reported the advantage of DIF in health sciences. Therefore, please discuss other related works in the discussion section. E.g.; Scott et al. Health Qual Life Outcomes. 2010 Aug 4;8:81. doi: 10.1186/1477-7525-8-811. Hagquist C. BMC Medical Research Methodology 19, 185 (2019). https://doi.org/10.1186/s12874-019-0828-32.\n\nResearch Ethics: Please provide an approved research ethics number for your study.\n\nPercentage (%) could be better representation rather than using actual number in Figure 2.\n\nIt would be better representation if authors invert color palette. Author is now using blue for positive and red for negative but I think blue for negative and red for positive is more familiar with.\n\nPlease omit following sentence. \"From the above analyses, we generated various graphs. However, due to space constraints, we present only those graphs related to characteristics of omics biomarkers (OBM construct).\"\n\nIn page 10 to page 11, authors stated as \" Figure 4c indicates a clear difference in TCC curves, albeit in small mar- gins, of DIF item responses between academia and industry groups. On the other hand, the left graph for all items displayed a much smaller difference than the right graph of Figure 4c.\" In my eyes, the results of all Items and DIF Items are similar.\n\nIn page 10 to page 11, authors stated as \" Figure 4c indicates a clear difference in TCC curves, albeit in small mar- gins, of DIF item responses between academia and industry groups. On the other hand, the left graph for all items displayed a much smaller difference than the right graph of Figure 4c.\" In my eyes, the results of all Items and DIF Items are similar.\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?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-782
https://f1000research.com/articles/9-779/v1	28 Jul 20	{ "type": "Research Article", "title": "Patient recruitment to a diabetic retinopathy screening trial through optimised patient information materials: an embedded study within a trial (SWAT)", "authors": [ "Rebecca Sheridan", "Peter Knapp", "Peter Bower", "Vichithranie Madurasinghe", "Deborah M Broadbent", "Lola Awoyale", "Amu Wang", "Tracy Moitt", "on behalf of the ISDR Trial Group", "Rebecca Sheridan", "Peter Bower", "Vichithranie Madurasinghe", "Deborah M Broadbent", "Lola Awoyale", "Amu Wang", "Tracy Moitt" ], "abstract": "Background: Printed participant information about trials is often technical, long and difficult to navigate. Optimisation and user testing can improve information materials, and may improve participant understanding and rates of recruitment. Methods: A study within a trial (SWAT) was undertaken within the ISDR trial. Potential participants in the ISDR trial were randomised to receive either the standard trial information or revised information that had been optimised through information design and user testing. Results: A total of 3,169 patients were randomised in the SWAT. Recruitment rates to the ISDR trial were 25.3% in the optimised information group and 26.1% in the standard information group (odds ratio 0.951; 95% CI 0.752 to 1.201; p=0.672). Clinic attendance rates were 71.6% in the optimised information group and 69.3% in the standard information group (OR 1.145; 95% CI 0.885 to 1.480; p=0.304). Conclusions: Optimisation of participant information through information design and user testing did not affect rate of recruitment to the host ISDR trial. Registration: ISRCTN ID ISRCTN87561257; registered on 08 May 2014.", "keywords": [ "SWAT", "trial", "recruitment", "patient information", "user testing", "diabetic retinopathy", "screening" ], "content": "Introduction\n\nInformation materials for potential randomised controlled trial participants are often long and complex1–3. This can result in a lack of understanding of key study details1,4,5, limiting the ability to provide informed consent.\n\nOne approach to improve materials is through optimisation and user testing, involving revisions to the text and design based on people’s ability to find and understand informational content6. Whilst people tend to prefer the materials revised after user testing7,8, a recent review concluded there was no evidence that optimised information materials improve recruitment9. However, the relevant evidence base is small10–14 and a recent ‘review of reviews’ found that information quality can facilitate research participation15.\n\n\nStudy aims\n\nThis study within a trial (SWAT) aimed to assess whether optimisation through user testing of patient information materials could increase recruitment to the Individualised Screening for Diabetic Retinopathy (ISDR) trial16.\n\n\nMethods\n\nISDR was approved by the Health Research Authority (REC reference: 14/NW/0034). The SWAT was approved by Yorkshire and the Humber REC – South Yorkshire (11/YH/0271). The REC waived the requirement to obtain participant consent for the SWAT.\n\nSWAT conducted within ISDR, which investigated the safety and acceptability of changing from annual screening to personalised (individualised) risk-based screening for diabetic patients16. This study is one of the SWATs run by the MRC-funded Systematic Techniques to Assist Recruitment to Trials (START) programme17.\n\nSWAT participants were eligible for ISDR18 and aged 16 years or older.\n\nAll participants were posted a study invitation letter and participant information sheet (PIS) alongside their annual screening clinic appointment. The control group received the standard ISDR materials (see Extended data19) whilst the intervention group were sent optimised patient information materials (see Extended data20) developed through two rounds of user testing.\n\nIf the patient attended their scheduled screening appointment, they were approached by a researcher to determine whether they had received, read and understood the information and whether they wanted to participate in ISDR. Clinic attendance and trial participation were recorded. If a researcher was not available on the clinic date, patients were not invited to participate.\n\nUser testing was undertaken face-to-face by Luto Research Limited at their premises in Leeds, UK, and involved 20 people, to reflect the age and gender distribution of the ISDR target population. In the first testing round 10 participants were given printed copies of materials and read the standard invitation letter and PIS (see Extended data)19. They were then asked to locate and demonstrate their understanding of 16 key items of trial information within the materials6. Materials were then revised based on participants’ responses. A second testing round was then completed using the same method, testing revised versions of the PIS and invitation letter.\n\nThrough testing, wording edits were made to the invitation letter to simplify content. Changes to the PIS included adding a title page, a summary of key points and a contents page, highlighting headings using coloured text and enlarged font, and simplifying wording. The final optimised PIS was presented as an A5 booklet (see Extended data)20.\n\nThe primary outcome measure was the proportion of patients in each group who were randomised within ISDR. The secondary outcome was the proportion of patients attending their screening appointment.\n\nA power estimate was generated using an estimated baseline recruitment rate of 20%, whereby running the trial for 16 weeks (clusters) would provide 84% power to detect a planned 10% difference (alpha 0.05).\n\nCluster randomised allocation to receive the standard or optimised PIS by week of mail-out (1:1), by random number generator, determined by date of clinic appointment; the SWAT ran for sixteen weeks (January-May 2016). Patients attended clinic at one of seven sites across Liverpool, UK. Concealment of allocation was achieved because the appointment schedule was set before SWAT allocations were randomised. Recruiting researchers were not masked as they saw the ISDR booklet the patients brought with them; patients were not masked but were nevertheless unaware that a SWAT was ongoing.\n\nOdds ratios (ORs) and corresponding 95% confidence intervals (CIs) were calculated to compare the proportion of patients from each randomised group (standard or optimised information) and the proportion of patients attending their appointment. Intention-to-treat analysis was used, with patients randomised to the SWAT irrelevant of whether a researcher was available for recruitment. Analyses were adjusted for cluster design and conducted in Stata version 14.221.\n\n\nResults\n\n3,169 participants were invited, 1,503 (47.4%) were randomised to the control group and 1,666 (52.6%) to the intervention group (Figure 1)22.\n\nA total of 2,235 (70.5%) patients attended a screening appointment and 815 (25.7%) patients were randomised to host trial (Table 1). There was no difference between the control group and the intervention group in randomisation (26.1% vs 25.3%; OR=0.951, 95% CI 0.752 to 1.201, p=0.672) or attendance (69.3% vs 71.6%; OR=1.145, 95% CI 0.885 to 1.480, p=0.304).\n\nAn additional 620 patients attended an appointment when no researcher was present and therefore were not asked to participate in ISDR. Sensitivity analysis including those patients did not substantially alter results.\n\n1 Intra-cluster correlation coefficient is 0.008.\n\n2 Intra-cluster correlation coefficient is 0.004.\n\n\nDiscussion\n\nThere was no statistically significant difference in randomisation to ISDR or attendance rates between those receiving standard or optimised materials. This is consistent with previous research9, including other embedded trials within MRC START which have observed only small effects on recruitment11–13.\n\nThere was no prior reason to expect recruitment rates to be affected by date of posting because choice of mail-out date was determined by clinic appointment and there were no systematic trends in appointments by time.\n\nWhilst there was no impact on recruitment, the optimised materials may have improved understanding of the trial thus enabling patients to make a more informed decision. Improved comprehension could also increase retention, due to greater understanding of the trial prior to recruitment. These outcomes were not assessed and further research examining this is warranted.\n\nThe study sample size was large, and results are likely to be generalisable to adult diabetic patients.\n\n\nConclusion\n\nOptimised patient information materials did not affect appointment attendance rates or randomisation to the host trial.\n\n\nData availability\n\nFigshare: ISDR trial SWAT dataset. https://doi.org/10.6084/m9.figshare.1238813622.\n\nFigshare: ISDR trial SWAT original participation materials. https://doi.org/10.6084/m9.figshare.12388190.v119.\n\nThis project contains the following extended data:\n\n- Appendix 1 – Original ISDR trial invitation letter.docx\n\n- Appendix 2 – Original ISDR trial PIS.docx\n\nFigshare: ISDR trial SWAT optimised participant materials. https://doi.org/10.6084/m9.figshare.12388220.v120.\n\nThis project contains the following extended data:\n\n- Appendix 3 – Optimised ISDR trial invitation letter.docx\n\n- Appendix 4 – Optimised ISDR PIS.pdf\n\nFigshare: CONSORT checklist for ‘Patient recruitment to a diabetic retinopathy screening trial through optimised patient information materials: an embedded study within a trial (SWAT)’. https://doi.org/10.6084/m9.figshare.12388175.v123.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nWe thank Luto Research Limited (luto.co.uk) for undertaking the user testing, and Making Sense (makingsense.co.uk) for graphic design input. The authors would also like to thank the participants in the embedded trial, the screening clinics involved in the SWAT, Jo Rick for her contributions to the early stages of this research, and the administrative support of Christopher Grierson, David Szmyt and Alannah Nightingale.\n\nThe ISDR Trial Group is: Simon P Harding (Chair), Deborah M Broadbent (ISDR Trial Principal Investigator), Paula Byrne, Anthony C Fisher, Mark Gabbay, Marta García-Fiñana, Marilyn James, Tracy Moitt, John R Roberts, Daniel Seddon, Irene M Stratton, Paula Williamson, Duncan Appelbe, Lola Howard, Ayesh Alshukri, Abigail Bennett, Christopher P Cheyne, Paula Byrne, Antonio Eleuteri, Christopher Grierson, Bryar Kadir, Mehrdad Mobayen-Rahni, Andrew Ovens, Christopher J Sampson, David Szmyt, Clare Thetford, Amu Wang, Helen Cooper, John Collins, Sue Howlin, John Kelly, Nathalie Massat, Gideon Smith, Vineeth Kumar, Chris Rogers, Julia West, Naveed Younis.\n\n\nReferences\n\nMontalvo W, Larson E: Participant comprehension of research for which they volunteer: a systematic review. J Nurs Scholarsh. 2014; 46(6): 423–31. PubMed Abstract \| Publisher Full Text\n\nManta CJ, Ortiz J, Moulton BW, et al.: From the Patient Perspective, Consent Forms Fall Short of Providing Information to Guide Decision Making. J Patient Saf. 2016. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLarson E, Foe G, Lally R: Reading level and length of written research consent forms. Clin Transl Sci. 2015; 8(4): 355–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFortun P, West J, Chalkley L, et al.: Recall of informed consent information by healthy volunteers in clinical trials. QJM. 2008; 101(8): 625–9. PubMed Abstract \| Publisher Full Text\n\nGriffin JM, Struve JK, Collins D, et al.: Long term clinical trials: how much information do participants retain from the informed consent process? Contemp Clin Trials. 2006; 27(5): 441–8. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAppelbe D, Broadbent D, Mobayen-Rahni M, et al.: Introducing personalised risk based intervals in screening for diabetic retinopathy: development, implementation and assessment of safety, cost-effectiveness and patient experience (ISDR): a case study in the use of automated systems in trials. Trials. 2015; 16(Suppl 2): O59. Publisher Full Text \| Free Full Text\n\nRick J, Graffy J, Knapp P, et al.: Systematic techniques for assisting recruitment to trials (START): study protocol for embedded, randomized controlled trials. Trials. 2014; 15: 407. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBroadbent DM, Sampson CJ, Wang A, et al.: Individualised screening for diabetic retinopathy: the ISDR study—rationale, design and methodology for a randomised controlled trial comparing annual and individualised risk-based variable-interval screening. BMJ Open. 2019; 9(6): e025788. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKnapp P, Sheridan R, Madhurasinghe V, et al.: ISDR trial SWAT original participation materials. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12388190.v1\n\nKnapp P, Sheridan R, Madhurasinghe V, et al.: ISDR trial SWAT optimised participant materials. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12388220.v1\n\nStataCorp: Stata Statistical Software: Release 14. College Station, TX: StataCorp LP. 2015. Reference Source\n\nKnapp P, Sheridan R, Madhurasinghe V, et al.: ISDR trial SWAT dataset. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12388136\n\nKnapp P, Sheridan R, Madhurasinghe V, et al.: ISDR trial SWAT CONSORT checklist. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12388175.v1" }	[ { "id": "83385", "date": "22 Apr 2021", "name": "Winfried M Amoaku", "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 on a study within a trial (SWAT) undertaken within the ‘Individualised Screening for Diabetic Retinopathy’ (ISDR) trial to assess optimisation through user testing of patient information materials, and effects on recruitment. This was to test a hypothesis that there was no evidence that optimised information materials improve recruitment into studies.\nPotential participants in the ISDR trial were randomised to receive either the standard trial information or revised information optimised through information design and user testing. Amongst the 3,169 randomised into this study recruitment rates were 25.3% in the optimised information group and 26.1% in the standard information group. Clinic attendance rates were similar in the study groups.\nThe study concluded that optimisation of participant information through information design and user testing did not affect rate of recruitment. The study findings are useful, and worth indexing.\n\nHowever, the manuscript can be improved with revisions as follows: ‘Introduction’ Para 1 line 3: to read’ ‘the ability of potential participants to…’ Para 2 line 2: to read: ‘which involves..’ (instead of ‘involving’) Para 2 line 6: change ‘improve’ to ‘improved’\n‘Intervention’ Revise to read: ‘All participants were sent study information……alongside their annual screening clinic appointment by post.’\n‘Results’ Avoid commencing sentences with digitised numbers. Insert ‘A total of’ preceding ‘3,169 participants’.\nUser Testing, Line 9: insert and suggestions after “participants’ responses” to read “participant responses and suggestions”.\n\nIs the work 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": "99962", "date": "26 Nov 2021", "name": "Manavi D Sindal", "expertise": [ "Reviewer Expertise Vitreoretinal surgery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe Authors present interesting information on optimised patient information material and its impact on patient recruitment to the trial.\n\nThey randomised patients into two arms- one arm received standard PIS, while the other received an optimised PIS with more details on the trial as well as simpler language. The optimised material was developed after mock rounds with age matched controls.\n\nThe SWAT revealed that the attendance to screening as well as recruitment to ISDR trial was comparable in both arms. Use of optimised material did not alter the attendance rates.\n\nThe authors utilise robust methodology and sound statistics.\n\nA few minor alterations can make the manuscript better:\nIn the abstract- ISDR needs expansion.\n\nIn sample size calculation- the derived sample size has to be mentioned.\n\nDiscussion can be more elaborate, and mention results of previous reports.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-779
https://f1000research.com/articles/9-778/v1	28 Jul 20	{ "type": "Case Report", "title": "Case Report: COVID-19 in a female patient who presented with acute lower limb ischemia", "authors": [ "Ahmed Muhi Fahad", "Ayam Ali Mohammad", "Hasanain A. Al-Khalidi", "Qusay Jummaa Lazim", "Fahad Ibrahim Hussein", "Ahmed Salih Alshewered", "Ahmed Muhi Fahad", "Ayam Ali Mohammad", "Hasanain A. Al-Khalidi", "Qusay Jummaa Lazim", "Fahad Ibrahim Hussein" ], "abstract": "Coronavirus disease 2019 (COVID-19) has developed as a pandemic and has caused thousands of deaths worldwide. It may be complicated with arterial or venous thrombosis; however, the literature for concerning these symptoms is limited. Here, we report a rare presentation of COVID-19 infection in a 49-year-old female patient, who presented with acute lower limb ischemia one day before the development of the classic symptoms for COVID-19, such as fever and dyspnea. Clinicians should have a high suspicion and awareness of COVID-19 infection in patients presenting with acute lower limb ischemia, especially during the pandemic period.", "keywords": [ "COVID-19", "Acute limb ischemia", "Arterial thrombosis", "Misan" ], "content": "Introduction\n\nCoronavirus disease 2019 (COVID-19) is a global pandemic disease caused by the SARS-COV-2 virus surface spike protein binding to the human angiotensin-converting enzyme 2 (ACE2) receptor, which is expressed in the lung (type 2 alveolar cells), heart, intestinal epithelium, vascular endothelium, and kidneys, providing a mechanism for multi-organ dysfunction. The median incubation period time is 4 to 5 days and 97.5% of patients will exert symptoms within 11.5 days1.\n\nTo the best of our knowledge, there is currently no research available in Iraq showing that ischemia of the lower limb caused by thrombosis is a rare presenting clinical feature of COVID-19. Here, we report a rare presentation of lower limb ischemia in a patient one day before the development of the classic symptoms for COVID-19, such as fever and dyspnea.\n\n\nCase report\n\nA 49-year-old female patient admitted to the emergency unit with severe agonizing left lower limb pain of one-day duration. No drug, medical, surgical, and smoking history was reported, but the patient did report contact with COVID-19 patients. On examination, the patient was a middle-aged woman, who seemed to be in discomfort. The left leg was cyanosed and discolored up-to the mid-leg, cold, with no detected dorsalis pedis or posterior tibial artery pulsations. There was popliteal artery pulsation, and no detected sensation in the whole left foot and distal third of leg without movement (Figure 1).\n\nNeurological, cardiac, abdominal, and locomotor system examinations were normal with mild crackles in the lung bases. Blood pressure on admission was 130/70 mmHg, heart rate was 123 beats/minute and the patient had a regular body temperature of 37.3°C. The patient’s respiratory rate was 18 breaths/minute and SPO2 was 75% on room oxygen. The patient underwent a blood test, electrocardiogram (ECG), Doppler study for lower limb arterial examination, and chest radiograph. The blood test results showed hemoglobin of 10.2 g/dl (normal range adult females, 12–16 g/dl), white blood cell count of 17200/ul (normal range, 4000–10000/ul), platelets of 847000/ul (normal range, 165000–415000/ul), lymphocytes of 2800/ul (normal range, 800–2600/ul), blood urea of 8 mg/dl (normal range, 15–45 mg/dl) and D-dimer of 2 (normal range, <0.5). Other investigations such as prothrombin time (PT), activated partial thromboplastin time (aPTT), international normalised ratio (INR) and liver enzymes were not available. The Doppler study showed normal flow in the left common femoral artery, superficial femoral artery, popliteal artery, and both proximal thirds of anterior tibial artery (ATA) and posterior tibial artery (PTA) with abrupt interruption of blood flow in the distal two-thirds of ATA and PTA. Chest X-ray showed large bilateral multiple lung shadows (Figure 2), and the ECG showed sinus tachycardia (Figure 3).\n\nThe patient was initiated with an anticoagulant – unfractionated heparin (therapeutic dose of 10,000 Units, in 100mL of 5% Dextrose intravenously (IV) every 6 hours) – fluid therapy (5% Dextrose 250 ml IV every 8 hours; 0.9% Sodium Chloride 250ml IV every 8 hours), oxygen therapy, pain killers (paracetamol injection; (1 g (10mg/100mL) IV every 8 hours), and ceftriaxone injection (1 g IV every 12 hours). The patient was then sent for a CT scan of the chest. This showed bilateral multiple opacities of the lung, suggesting a suspicion of COVID-19 infection (Figure 4). Consequently, the patient was sent to a COVID-19 isolation ward, and nasal and throat PCR test for COVID-19 was taken. Three days post-admission, the COVID-19 test returned a positive result.\n\nOn the second day post-admission, the patient developed clear dyspnea and tachypnea. Her saturation dropped from 93% to 60% (normal pulse oximeter readings range from 95–100%, values under 90% are considered low); therefore, the patient was intubated. The left lower limb was non-viable up to the mid-leg, meaning that the patient was a good candidate for amputation. However, the patient was still unstable and unfit for such a surgery. On the fourth day post-admission, the patient developed sudden cardiac arrest due to persistent hypoxia and unfortunately died.\n\n\nDiscussion\n\nIn severe cases, COVID-19 infection can develop disseminated intravascular coagulopathy with fulminant activation of coagulation leading to widespread microvascular thrombosis2. This may be reflected by high D-dimer, prolongation of PT, aPTT, INR and decreased fibrinogen levels, which are not available in our center3. Our case shows that one should keep in mind that acute limb ischemia may be a clinical feature of COVID-19 infection as an isolated early symptom, in addition to other features, or may develop as a complication of disease during the admission period due to intravascular thrombosis2. In our patient, as in many cases of acute lower limb ischemia, the diagnosis was early as it depended on clinical history and examination with Doppler study.\n\nOur case differs from two case reports by Kaur et al. In the first, the patient was a 43-year-old man with a history of hypertension and diabetes mellitus, who presented with acute lower limb ischemia and later developed a fever and exertional dyspnea3. The second reported a 71-year-old Hispanic male with a history of diabetes mellitus who presented with fever, dry cough and exertional dyspnea followed by development of sudden onset severe upper right arm pain diagnosed as intraluminal thrombosis of the axillary artery4. Our patient had no comorbidities and presented with acute lower limb ischemia as an early feature to COVID-19 infection before the development of fever or other respiratory features. In addition, our patient did not undergo surgery due to her instability and metabolic derangement, while in the second report by Kaur et al, the patient underwent thromboembolectomy of the axillary artery4. In a study by Bellosta et al, revascularization was done in 17 out of 20 patients, but this was successful in 12 patients only2. Unfortunately, our patient died due to cardiac arrest by persistent hypoxia.\n\nWe present an unusual presentation of COVID-19 infection as acute lower limb ischemia in a female patient without comorbidities. Doctors should have a high suspicion of COVID-19 infection in such a case especially during the pandemic period.\n\n\nConsent\n\nWritten informed consent for the publication of the article and any associated images was obtained from the patient before she died. Permission to publish was also sought from the patient’s husband after the patient died.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nClerkin KJ, Fried JA, Raikhelkar J, et al.: COVID-19 and Cardiovascular Disease. Circulation. 2020; 141(20): 1648–1655. PubMed Abstract \| Publisher Full Text\n\nBellosta R, Luzzani L, Natalini G, et al.: Acute limb ischemia in patients with COVID-19 pneumonia. J Vasc Surg. 2020; S0741-5214(20)31080-6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaur P, Qaqa F, Ramahi A, et al.: Acute upper limb ischemia in a patient with COVID-19. Hematol Oncol Stem Cell Ther. 2020; S1658-3876(20)30096-0. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKaur P, Posimreddy S, Singh B, et al.: COVID-19 Presenting as Acute Limb Ischaemia. Eur J Case Rep Intern Med. 2020; 7(6): 001724. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "68297", "date": "10 Aug 2020", "name": "Parminder Kaur", "expertise": [ "Reviewer Expertise Internal Medicine / Cardiology." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a interesting case report of unusual and life threatening presentation of COVID -19. However, it needs proofreading.\ntitle should be rephrased.\n\nabstract -- 2 nd line -- needs to be edited -- these concerning symptoms.\n\nkeywords -- misan-- unclear what is meant by it.\n\nIntroduction -- SARS-COV-2 -fullform.\n\nIntroduction -- first line needs to be edited --- like can be split into two sentences. first line --Coronavirus disease 2019 (COVID-19) is a global pandemic disease caused by the SARS-COV-2 virus. second line-- SARS-COV-2 virus binds to angiotensin-converting enzyme 2 (ACE2) receptor, which is expressed in the lung (type 2 alveolar cells), heart, intestinal epithelium, vascular endothelium, and kidneys, providing a mechanism for multi-organ dysfunction.\n\nchange 97.5% of patients will exert symptoms within 11.5 days-- to --- 97.5% of patients are symptomatic within 11.5 days.\n\nrephrase -- To the best of our knowledge, there is limited data available in Iraq regarding arterial thrombosis leading to acute limb ischemia in a COVID-19 patient.\n\ncase report part needs to be edited extensively -- pertinent exam needs to summarized in one line and needs to been order.\n\nskip this line-- The patient underwent a blood test, electrocardiogram (ECG), Doppler study for lower limb arterial examination, and chest radiograph.\n\nImaging studies should be written together and in order -- chest x ray/ ct scan / doppler followed by management and outcome.\n\nThe left lower limb was non-viable up to the mid-leg, meaning that the patient was a good candidate for amputation. However, the patient was still unstable and unfit for such a surgery ---- both these lines need to be edited / rewritten --\n\nOur patient had no comorbidities and presented with acute lower limb ischemia as an early feature to COVID-19 infection before the development of fever or other respiratory features--- CHANGE TO Our patient had no comorbidities and presented with acute lower limb ischemia as an early feature of COVID-19 infection before the development of classic respiratory symptoms.\n\nIn addition, our patient did not undergo surgery due to her instability and metabolic derangement,--specify what metabolic derangement / was patient hemodynamically unstable?\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": "68449", "date": "12 Aug 2020", "name": "Deepak Vedamurthy", "expertise": [ "Reviewer Expertise health outcomes research", "health care quality and safety", "served as judge for clinical vignettes for American College of Physicians and Society of Hospital Medicine." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA 49 yr old lady, non-smoker with apparently no significant medical problems presented to emergency room with acute left lower limb ischemia. She was normotensive, tachycardic, and hypoxic but with normal respiratory rate on presentation. She was anemic with leucocytosis (no lymphopenia), normal renal function (? creatinine) and elevated D-dimer. Rest of coagulation panel not available. PCR for COVID-19 positive. CT chest suggestive of COVID-19. On day 2, patient is intubated and day 4, patient dies.\n\nVenous thromboembolism is well described now in COVID 19 and high intensity DVT (deep vein thrombosis) prophylaxis is often recommended but arterial thromboembolism is not that common and effective prophylaxis has not been described. This make this a valuable case report and the authors do a good job of presenting an important and devastating manifestation of this important disease.\nHere are some additional areas that if clarified will help readers.\n\nPlease clarify what is “clear dyspnea” on physical exam.\n\nWas there any history of oral contraceptive usage?\n\nWas her blood group known? Recent genome wide association studies reveal this may be important\n\nWhat test (? Platform) was employed to confirm SARS-CoV2\n\nWhat was the date of presentation for patient- Was steroid, Plaquenil, tocilizumab or convalescent plasma treatment option offered? This is a disease with rapidly evolving treatment strategies.\n\nDid echocardiogram or did CT chest report any concern for LV thrombus.\n\nWas CT angiogram lower extremity performed?\n\nWas the patient also screened for DVT and Pulmonary embolism?\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? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "6132", "date": "23 Nov 2020", "name": "Ahmed muhi Fahad", "role": "Author Response", "response": "1- clear dyspnea means obvious shortness of breath 2- no history of OCP 3- no it is unknown 4- RT pCR 5- it is same day of admission , no treatment strategy was given before presentation 6-no portable echo was available in covid emergency era 7-pt unfit for transfer to CT angio 8- yes we did" } ] }, { "id": "72267", "date": "26 Oct 2020", "name": "Gabriele Piffaretti", "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\nThese Authors experienced what we faced on a daily basis during the outbreak of the pandemic in northern Italy. ALI was rapidly acknowledged as one of the potential presenting symptoms of the CV19 infection, a circumstance that soon we understood should have been treated aggressively with extensive surgery and iv heparin. This is a single case report: I cannot understand why this patient was not treated immediately owing to the diagnosis of ALI. Moreover, we have no further information regarding the relationship of the CV19 viral infection and the ALI event at least in terms of impact on patient's survival.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "6131", "date": "23 Nov 2020", "name": "Ahmed muhi Fahad", "role": "Author Response", "response": "Yes , as we diagnosed the case as ALI and we can not proceed for surgery , because the patient was unstable and unfit for surgery with distal arterial involvement and there is no intraarterial thrombolytic availability in our center with rapid deterioration of her limb . These are the causes behind the choice of conservative treatment" } ] } ]	1	https://f1000research.com/articles/9-778
https://f1000research.com/articles/8-562/v1	26 Apr 19	{ "type": "Research Article", "title": "HIV, syphilis and hepatitis B coinfections in Mkushi, Zambia: a cross-sectional study", "authors": [ "Cibangu Katamba", "Theresa Chungu", "Chisali Lusale", "Theresa Chungu", "Chisali Lusale" ], "abstract": "Background: Human immunodeficiency virus, syphilis and hepatitis B virus (HBV) are major global public health problems. They are sexually transmitted diseases with overlapping modes of transmission and affected populations. The aim of this study is to assess the seroprevalence of HIV 1, hepatitis B virus and syphilis coinfections among newly diagnosed HIV individuals aged 16 to 65 years, initiating on antiretroviral therapy, in Mkushi, Zambia. Methods: A total number of 126 sera were collected from HIV 1 infected patients attending Mkushi district hospital/ART clinic for antiretroviral therapy initiation. Hepatitis B surface antigen test and serologic test for syphilis were conducted between March and May 2018. Results: Of the 126 participants, hepatitis B surface antigen (HBsAg) was detected with a prevalence of 9.5% among newly diagnosed HIV infected patients, while that of syphilis was as high as 40.5% in this same population group. Three patients recorded HIV coinfections with both syphilis and hepatitis B virus (2.4%) at the same time. After analysis, the results indicate that there was no significant association between gender for both dependent variables: HIV/syphilis or HIV/hepatitis B virus coinfections (alpha significance level > 0.05). Those who had a history of syphilis infection in the past were more likely than those who had none to be HIV-syphilis coinfected (53.6% vs 34%, respectively; odd ratio [OR] 2.236; 95% confidence interval [CI] 1.045 – 4.782). Conclusion: The high prevalence rates for HIV, HBV, and syphilis coinfections strongly indicate the need for HBV and syphilis screening for HIV infected individuals. Furthermore, the high number of patients previously treated for syphilis who retest positive for syphilis in this study calls for use of the Venereal Disease Research Laboratory test to identify true syphilis infection (titers ≥ 1:8 dilutions, strongly suggestive).", "keywords": [ "HIV", "Hepatitis B", "Syphilis", "Coinfections" ], "content": "Introduction\n\nHepatitis B virus, together with syphilis and human immunodeficiency virus (HIV), are important public health challenges worldwide1, they are transmitted through sex (Sexual transmitted diseases, STDs)2–5 and cross over affected populations1. The World Health Organization (WHO) reported an estimated 36.9 million people were living with HIV in 20166,7. It was also reported that 248 million had chronic Hepatitis B virus (HBV) infection (persistent HBs Ag ≥ six months)8.\n\nIt is estimated that about a million sexually transmitted diseases are acquired daily throughout the world5. Moreover, STDs have impact on neonatal, sexual and reproductive health. They can also cause severe complications if not treated. Additionally, STDs pose major a socioeconomic challenge in the form of treatment costs5,9,10. Many studies have demonstrated a two-sided relationship between HIV and a number of STDs, including syphilis and HBV4. Syphilis and HBV has the potential to increase genital and plasma HIV 1 RNA levels. This increase can increase the likelihood of transmission of HIV 1 to others. In the same way, HIV 1 infection to impact the clinical manifestation, the therapeutic outcome and the HBV/syphilis disease progression4.\n\nHIV is associated with a higher prevalence of both HBV and Hepatitis C Virus (HCV) in Sub-Saharan Africa11–13 in this region, many people living with HIV are HBV or HCV coinfected. About 8% of individuals infected with HIV were HBV coinfected in another study conducted in this region14. In a trial conducted in Sub-Saharan Africa15, a number of factors were associated with high rates of HIV and syphilis coinfection, including: young age, divorce, widowhood, or separation. Although it is helpful to recognize syphilis risk factors, many women without the risk factor characteristics were syphilis-seroreactive15. Also, a high proportion of prevalent rapid plasminogen reagin (RPR)-positive infections remain serofast despite treatment16.\n\nIn a number of studies conducted in Zambia17–29, it has been observed that HIV and chronic hepatitis B (CHB) coinfection was common in both children and adults. It was also observed in these patients that many risk factors such as liver complications and impaired immunologic recovery increased morbidity and mortality. In these settings, syphilis infection remains prevalent in HIV infected adults30. As HIV, syphilis and HBV coinfections are clinically consequential, there is great need to screen for syphilis and HBV in people living with HIV/AIDS. The aim of this study was to determine the prevalence of HIV1, HBV and Treponema pallidum serological markers, and therefore their coinfection prevalence among newly diagnosed HIV individuals attending ART clinic in Mkushi rural district of Zambia.\n\n\nMethods\n\nThis study was approved by Mkushi District Health Office. Participants were informed and provided verbal consent prior to enrollment in the study. Samples were collected as part of routine recommended national baseline tests before initiating ART, therefore only verbal consent was sought.\n\nWe performed a cross-sectional study to assess the seroprevalence of HIV 1, HBV and syphilis coinfections among newly diagnosed HIV individuals aged 16 to 65 years, initiating on cART, in Mkushi, Zambia. Sociodemographic and clinical data were collected from study subjects including age, sex, number of years of education, marital status, residence, occupation, household income, HIV transmission route, history of tuberculosis, past syphilis infection, and WHO clinical staging. A data collection sheet (Extended data31) was used to collect information in addition to client’ files.\n\nInclusion and exclusion criteria: this study included a patient population starting ART between March and May 2018, aged 16 to 65 years old, confirmed HIV 1 infection, being ART naïve (never taken ART for their HIV infection), regardless of WHO clinical staging. Subjects previously exposed to ART, children below the age of 16, and clients with HIV 2 or HIV 1 & 2 coinfections were excluded. A total number of 126 subjects were included.\n\nDiagnosis: All tests were performed at Mkushi hospital laboratory. Four milliliters of venous blood were collected from each client using aseptic technic. Following centrifugation, sera were separated and tested immediately for HIV antibodies. HIV serologic test was conducted using Determine screening test (ALERE) (CHASE BUFFER REF 7D2243, ALERE Medical Co. Ltd. Japan); followed by SD Bioline confirmatory test, discriminating between HIV 1 & HIV 2. (SD HIV1/2 3.0) (Assay diluent Lot 03ADDC013, SD Standard diagnostics, Inc. Hepatitis B virus infection was assessed by an immunochromatographic test (one step rapid test, Lot No. HBPDR00117, NeckLife, Punjab), following the manufacturer’s instructions. Syphilis serology was examined using rapid plasma reagin (RPR) (one Step Rapid Test, Lot No. SYS-02-18Q, Ensure Biotech, Hyderabad-500 076), following the manufacturer’s instructions.\n\nStatistical analysis: The collected data were analyzed using the IBM SPSS statistics software (trial version 20). The Chi-Square test was used for analysis of the differences between proportions and p < 0.05 was considered as significant. The statistical analysis of frequencies was also conducted and the confidence interval was set at 95%.\n\n\nResults\n\nOf the 126 participants, 46 were male (36.5%) and 80 were female (63.5%), 102 were between the age of 19 and 40 years excluded (81%), 100 had primary education (79.4%). While 66 patients were married (52.4%), the rest were either divorced (30/126), never married (22/126) or widowed (8/126).\n\nA total number of 126 blood samples were collected from HIV 1 infected patients attending Mkushi district hospital/ART clinic for antiretroviral therapy initiation (see underlying data24). HIV 1, HBV, and Syphilis coinfections were recorded together in 2.4% of cases (3/126).\n\nTable 1 shows the prevalence of syphilis coinfection with HIV by age group.\n\nThe overall syphilis seroprevalence was 40.5% (51/126) in the studied population. HIV infected patients in the age group 20 – 29 years recorded the highest syphilis prevalence 47% (24/51), followed by the age group 30 – 39 years at 29% (15/51), while those in the age group below 20 years recorded the lowest prevalence of 2% (1/51).\n\nThe below table shows the hepatitis B virus and syphilis coinfections results by gender among HIV infected subjects (Table 2).\n\nHepatitis B surface antigen (HBsAg) was detected with a prevalence of 9.5% (12/126) among newly diagnosed HIV infected patients, while that of syphilis was as high as 40.5% (51/126) in this same population group. The above data was analyzed and the results indicate that there was no significant association between gender and syphilis (Chi square value = 0.373, p = 0.542). This study also shows that HBV presence was independent of gender (alpha significance level > 0.05).\n\nA past medical history was taken from each individual to obtain information on past syphilis infection. The results of past and current syphilis are shown in the table below (Table 3).\n\nFinally, those HIV clients who had a history of syphilis infection in the past were more likely than those who had none to be HIV-syphilis coinfected (53.6% vs 34%, respectively; OR 2.236; 95% CI 1.045 – 4.782). The relative Odds of HIV-Syphilis coinfection is calculated at 1.573 (95% CI: 1.044 – 2.370).\n\n\nDiscussion and conclusion\n\nThe correlates of HIV, syphilis and hepatitis B coinfections were age (between 20 and 39 years), primary education, and of course sexual activity. This may be influenced by the rural settings where our study was conducted (especially low level of education). The HBV/HIV coinfections prevalence of 9.5% in this study is in line with the prevalence of 9.9% documented in Zambia by Kapembwa and colleagues27, and also the 12.2 reported by Vinikoor et al.19. The later also demonstrated that HIV coinfected adult males were more likely to be coinfected with HBV than their female counterparts. This contrast to our findings may be due to our small sample size. In our study, Pearson Chi-square analysis showed no statistically significant difference between gender for both HIV/HBV and HIV/Syphilis coinfections. Our findings demonstrate that HIV infected clients are more likely to be infected with other STIs, especially syphilis. The coinfection prevalence of 40.5% for HIV and syphilis agrees with previous study conducted in Zambia (43%) by Odom and colleagues16. We found past syphilis infection to have positive association with two-fold increase risk of HIV/syphilis coinfections.\n\nCONCLUSION: The high prevalence rates for HIV, HBV, and syphilis coinfections strongly indicate the need for HBV and syphilis screening for HIV infected individuals. The 2018 Zambian consolidated guidelines for prevention and treatment of HIV infection to address the management of these coinfections32. However, there is urgent need to monitor the implementation of HBV and syphilis testing among HIV infected subject to close the gap between policy and practice. Furthermore, in previous study conducted in Zambia, Odom and colleagues16 demonstrated that a high proportion of RPR positive infections remain serofast despite treatment. The high number of patients previously treated for syphilis who retest positive for syphilis in this study calls for use of the Venereal Disease Research Laboratory test to identify true syphilis infection (titers ≥ 1:8 dilutions, strongly suggestive)33. Both government and Non-governmental organizations need to up their efforts to support these health care service needs appropriately.\n\n\nData availability\n\nHarvard Dataverse: Replication Data for: HIV, SYPHILIS AND HEPATITIS B COINFECTIONS IN MKUSHI RURAL, ZAMBIA: a cross-sectional study. https://doi.org/10.7910/DVN/AK3JZR34\n\nThis project contains the following underlying data:\n\nHIV_HBV_SYPHILIS.tab (Seroprevalence data)\n\nHarvard Dataverse: Cibangu, Katamba, 2019, \"Replication Data for: HIV, SYPHILIS AND HEPATITIS B COINFECTIONS IN MKUSHI RURAL, ZAMBIA: a cross-sectional study\". https://doi.org/10.7910/DVN/6SNMQS31\n\nThis project contains the following extended data:\n\nData collection sheet.docx (data collection sheet)", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nWe thank the management of Mkushi district hospital, the ART staff, and the laboratory personnel for their cooperation in facilitating this investigation.\n\n\nReferences\n\nEasterbrook PJ, Roberts T, Sands A, et al.: Diagnosis of viral hepatitis. Curr Opin HIV AIDS. 2017; 12(3): 302–314. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMutagoma M, Nyirazinyoye L, Sebuhoro D, et al.: Syphilis and HIV prevalence and associated factors to their co-infection, hepatitis B and hepatitis C viruses prevalence among female sex workers in Rwanda. BMC Infect Dis. 2017; 17(1): 525. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThio CL: Hepatitis B and human immunodeficiency virus coinfection. Hepatology. 2009; 49(5 Suppl): S138–45. PubMed Abstract \| Publisher Full Text\n\nChun HM, Carpenter RJ, Macalino GE, et al.: The Role of Sexually Transmitted Infections in HIV-1 Progression: A Comprehensive Review of the Literature. J Sex Transm Dis. 2013; 2013: 176459, 15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCarmona-Gutierrez D, Kainz K, Madeo F: Sexually transmitted infections: old foes on the rise. Microb Cell. 2016; 3(9): 361–362. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization: Global health sector response to HIV 2000–2015: focus on innovations in Africa: progress report. Geneva: World Health Organization; 2016. Reference Source\n\nStover J, Andreev K, Slaymaker E, et al.: Updates to the spectrum model to estimate key HIV indicators for adults and children. AIDS. 2014; 28 Suppl 4: S427–S434. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchweitzer A, Horn J, Mikolajczyk RT, et al.: Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013. Lancet. 2015; 386(10003): 1546–1555. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Draft global health sector strategies: Sexually transmitted infections, 2016–2021. 2015. Reference Source\n\nWorld Health Organization: Sexually Transmitted Infections (STIs). 2015. Reference Source\n\nBarth RE, Huijgen Q, Taljaard J, et al.: Hepatitis B/C and HIV in sub-Saharan Africa: an association between highly prevalent infectious diseases. A systematic review and meta-analysis. Int J Infect Dis. 2010; 14(12): e1024–e1031. PubMed Abstract \| Publisher Full Text\n\nMoges F, Kebede Y, Kassu A: Seroprevalence of HIV, Hepatitis B infections and syphilis among street dwellers in Gondar city, Northwest Ethiopia. Ethiop J Health Dev. 2006; 20(3). Publisher Full Text\n\nShimelis T, Tassachew Y, Tadewos A, et al.: Coinfections with hepatitis B and C virus and syphilis among HIV-infected clients in Southern Ethiopia: a cross-sectional study. HIV AIDS (Auckl). 2017; 9: 203–210. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCoffie PA, Egger M, Vinikoor MJ, et al.: Trends in hepatitis B virus testing practices and management in HIV clinics across sub-Saharan Africa. BMC Infect Dis. 2017; 17(Suppl 1): 706. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPotter D, Goldenberg RL, Read JS, et al.: Correlates of Syphilis Seroreactivity Among Pregnant Women: The HIVNET 024 Trial in Malawi, Tanzania, and Zambia. NIH Public Access. Sex Transm Dis. 2006; 33(10): 604–609. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDionne-Odom J, Karita E, Kilembe W, et al.: Syphilis treatment response among HIV-discordant couples in Zambia and Rwanda. Clin Infect Dis. Oxford University Press on behalf of the Infectious Diseases Society of America. 2013; 56(12): 1829–37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeebles K, Nchimba L, Chilengi R, et al.: Pediatric HIV-HBV Coinfection in Lusaka, Zambia: Prevalence and Short-Term Treatment Outcomes. J Trop Pediatr. 2015; 61(6): 464–467. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVinikoor MJ, Sinkala E, Mweemba A, et al.: Elevated AST-to-platelet ratio index is associated with increased all-cause mortality among HIV-infected adults in Zambia. HHS Public Access. Liver Int. 2015; 35(7): 1886–1892. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVinikoor MJ, Musukuma K, Munamunungu V, et al.: Implementation of routine screening for chronic hepatitis B virus co-infection by HIV clinics in Lusaka, Zambia. HHS Public Access. J Viral Hepat. 2015; 22(10): 858–860. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYager P, Domingo GJ, Gerdes J: Point-of-care diagnostics for global health. Annu Rev Biomed Eng. 2008; 10: 107–44. PubMed Abstract \| Publisher Full Text\n\nVinikoor MJ, Mulenga L, Siyunda A, et al.: Association between hepatitis B co-infection and elevated liver stiffness among HIV-infected adults in Lusaka, Zambia. HHS Public Access. Trop Med Int Health. 2016; 21(11): 1435–1441. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPang T, Peeling RW: Diagnostic tests for infectious diseases in the developing world: two sides of the coin. Trans R Soc Trop Med Hyg. 2007; 101(9): 856–7. PubMed Abstract \| Publisher Full Text\n\nChisenga CC, Musukuma K, Chilengi R, et al.: Field performance of the Determine HBsAg point-of-care test for diagnosis of hepatitis B virus co-infection among HIV patients in Zambia. HHS Public Access. J Clin Virol. 2018; 98: 5–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoffmann CJ, Charalambous S, Thio CL, et al.: Hepatotoxicity in an African antiretroviral therapy cohort: the effect of tuberculosis and hepatitis B. AIDS. 2007; 21(10): 1301–8. PubMed Abstract \| Publisher Full Text\n\nWandeler G, Musukuma K, Zürcher S, et al.: Hepatitis B Infection, Viral Load and Resistance in HIV-Infected Patients in Mozambique and Zambia. PLoS One. 2016; 11(3): e0152043. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWandeler G, Gsponer T, Bihl F, et al.: Hepatitis B virus infection is associated with impaired immunological recovery during antiretroviral therapy in the Swiss HIV cohort study. J Infect Dis. 2013; 208(9): 1454–8. PubMed Abstract \| Publisher Full Text\n\nKapembwa KC, Goldman JD, Lakhi S, et al.: HIV, Hepatitis B, and Hepatitis C in Zambia. J Glob Infect Dis. 2011; 3(3): 269–274. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHawkins C, Christian B, Ye J, et al.: Prevalence of hepatitis B co-infection and response to antiretroviral therapy among HIV-infected patients in Tanzania. AIDS. 2013; 27(6): 919–27. PubMed Abstract \| Publisher Full Text\n\nKasolo F, Sakala I, Baboo K: Paris, France: Conference on HIV Pathogenesis and Treatment; Hepatitis B virus infection in human immunodeficiency virus seropositive patients at the University Teaching Hospital, Lusaka, Zambia: Interrelationship. [Abstract no. 963]. 2nd. 2003.\n\nMakasa M, Fylkesnes K, Michelo C, et al.: Declining syphilis trends in concurrence with HIV declines among pregnant women in Zambia: observations over 14 years of national surveillance. Sex Transm Dis. 2012; 39(3): 173–81. PubMed Abstract \| Publisher Full Text\n\nKatamba C: Replication Data for: HIV, SYPHILIS AND HEPATITIS B COINFECTIONS IN MKUSHI RURAL, ZAMBIA: a cross-sectional study. Harvard Dataverse, V1. 2019. http://www.doi.org/10.7910/DVN/6SNMQS\n\nZambia Consolidated Guidelines for prevention and treatment of HIV infection. Republic of Zambia, Ministry of Health. 2018. Reference Source\n\nWorld Health Organization: Sexually transmitted and other reproductive tract infections: a guide to essential practice. Geneva: World Health Organization; 2005. Reference Source\n\nKatamba C: Replication Data for: HIV, SYPHILIS AND HEPATITIS B COINFECTIONS IN MKUSHI RURAL, ZAMBIA: a cross-sectional study. Harvard Dataverse, V1. 2019. http://www.doi.org/10.7910/DVN/AK3JZR" }	[ { "id": "58393", "date": "03 Feb 2020", "name": "Sanika Chirwa", "expertise": [ "Reviewer Expertise Neuroscience & Clinical Pharmacologist - actively engaged in both basic and clinical research. Later includes focus on patient-centered care of HIV continuum of care with a focus on pediatric HIV." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 great potential in the presented study in terms of highlighting HIV + HBV + syphilis comorbid profile within rural settings in Zambia. This information could guide the distribution of pertinent resources needed to curtail the infections while identifying gaps in the drive to end the HIV epidemic. However, the manuscript is not written to best advantage and will require significant revisions to be impactful. Within this context, the authors may wish to consider the following concerns.\n\nINTRODUCTION\nThis has accurate information but fails to specifically indicate relevance and need for study. What are the current prevalence estimates for HIV/HBV and/or syphilis co-infections in Mkushi? in Zambia? Is this information lacking and, therefore, partly formed the basis for the research study?\n\nAre laboratory-based screening and/or diagnostic tests for the three infections routinely conducted together in much of Zambia, or this is lacking and provided another impetus for the study?\n\nMETHODS\nClarify if the “Mkushi District Health Office” has formal authority to independently review and give ethical approval to conduct clinical research studies consistent with the norm.\n\nIndicate how study participants were informed of study, and how (and who) conducted the enrollment into the study.\n\nState what percentage of new HIV cases initiated on cART did the reported 126 patients (i.e., enrolled in study between March and May 2018) reflect.\n\nIt is unclear how many 4-mL venous blood draws were carried out on each patient. Were these blood draws separate from routine blood draws for patient care at the hospital?\n\nProvide more information on how the screening/diagnostics tests performed actually work. Indicate their limitations and/or likelihood of false-positives. For example, Rapid Plasma Reagin for syphilis detects nonspecific antibodies produced by the body while fighting other infections like Lyme disease. How was this checked in study?\n\nRESULTS\nPresented data are scanty and lack details. The authors are encouraged to expand this section and give additional information as well as correct errors in counts.\n\nConsider reporting socioeconomic and demographic characteristics of participants in Table format. Give values as mean ± STD here and elsewhere, accordingly.\n\nStated syphilis seroprevalence is 51 of 126 cases. This is then subdivided into age groups, i.e., 1 for below 20 years; 24 for 20–29 years; and 15 for 30 – 39 years. But this yields a total of 40 and not 51 cases. What happened to the missing 11 patients?\n\nLike with syphilis, should also report “seroprevalence” data for HIV and HBV both by age and gender. Authors need to be consistent accordingly.\n\nNeed clarification on the reliability of the collection of “past syphilis infection” history. How exactly was this done?\n\nMore rigorous statistical analyses needed to properly interpret data and show significance as applicable. The significance of findings are unconvincing (and lost) as reported.\n\nDISCUSSION\nInterpretation and significance of findings are unconvincing. It is difficult to make an independent assessment since presented data lack much detail.\n\nNeed to explain what gaps in knowledge have been filled by the study. For example, how will this information assist with patient care and/or re-direction of resources for concomitant screenings/diagnosis of HIV, HBV, and syphilis?\n\nAre the above services readily available in all health centers in Zambia, or there is a disparity between urban and rural health centers? Explain.\n\nNeed to discuss the limitations of the research study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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? Partly", "responses": [ { "c_id": "5688", "date": "27 Jul 2020", "name": "Cibangu Katamba", "role": "Author Response", "response": "Thank you very much for reviewing this manuscript. The relevance and need for the study have been included. The number of participants informed of the study and how/ who conducted the study have been described. The 4 Ml venous blood draws were the routine blood draws for ART clients base line tests as recommended by the national guidelines. Limitation of screening diagnostic tests have been described. Data errors have been corrected. The total number of patients who were seen during this period has also been included as the enrollment. Citations have been checked and corrected accordingly. The waiver for ethical clearance was sought and obtained from the ERES Converge Zambian Institutional Review Board (IRB), the authority to publish was sought and obtained from the Zambian National Health Research Authority (NHRA). This will also appear in the new version. The limitations of the research have also been discussed." } ] }, { "id": "64070", "date": "23 Jun 2020", "name": "Carolyn Bolton Moore", "expertise": [ "Reviewer Expertise Infectious Disease in Zambia. Paediatric HIV. 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\nThank you for the opportunity to review this manuscript. Overall the manuscript is sound but there are several grammatical errors and some sentences that do not make sense eg the reference to the National Guidelines. Additionally, there does not seem to be ethical approval. My understanding is that Mkushi District Health Office is the administrative body that falls under the Ministry of Health and manages the public health services in that area. Unless they have a special mandate, of which I am not aware, they are not authorized to provide Ethical Clearance as they are not considered a regulatory body. They are, however, required to give permission for research to occur in their facilities. Given that this study involves human subjects, I recommend getting retrospective ethics approval if not yet obtained or, if it has been obtained, including this in the manuscript from a recognised regulatory authority. Also, please include the total number of patients who were seen during the same period as the enrolment (ie the denominator), it is not clear what proportion of patients who were seen, enrolled in the study and had a specimen drawn. Please also check citations- the paragraph that starts with ... in Zambia (17-29) includes references that are regional but not necessarily in Zambia. Lastly, I suggest using STI instead of STD as it appears many of these infections were subclinical.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5687", "date": "27 Jul 2020", "name": "Cibangu Katamba", "role": "Author Response", "response": "Thank you very much for reviewing this manuscript. The grammatical errors have been revised and the new version will be availed soon. Waiver for ethical clearance was sought and obtained from the ERES Converge Zambian Institutional Review Board (IRB), the authority to publish was sought and obtained from the Zambian National Health Research Authority (NHRA). This will also appear in the new version. The total number of patients who were seen during this period has also been included as the enrollment. Citations have been checked and corrected accordingly. The term STI has been used instead of STD." } ] } ]	1	https://f1000research.com/articles/8-562
https://f1000research.com/articles/8-2105/v1	16 Dec 19	{ "type": "Research Article", "title": "The effect of the addition of propolis to resin modified glass ionomer cement bracket adhesive materials on the growth inhibition zone of Streptococcus mutans", "authors": [ "Stefani Kristanti Saputra", "Darmawan Sutantyo", "Cendrawasih Andusyana Farmasyanti", "Ananto Ali Alhasyimi", "Darmawan Sutantyo", "Ananto Ali Alhasyimi" ], "abstract": "Background: Orthodontic treatments progress alongside the development of adhesive materials. The aim of the present study was to determine the antibacterial properties of propolis, a natural product, in a mixture of resin modified glass ionomer cement by observing the growth inhibition zone of Streptococcus mutans. Methods: This was an in vitro study conducted on 45 samples of adhesive material, which were divided into three groups of propolis concentrations (0%, 15%, and 25%) and duration (0, 15, and 30 days). The antibacterial effect of each sample was evaluated against S. mutans using an agar plate diffusion test. Measurement of the diameter of the growth inhibition zone of S. mutans were carried out. The data obtained were analyzed statistically by Kruskal Wallis test. Results: There was a relationship between concentration and duration of propolis to the growth inhibition zone of S. mutans (p<0.05). The addition of 25% propolis concentration inhibited the growth of S. mutans more than the addition of 15% and 0% propolis concentration. The addition of 0%, 15%, and 25% propolis concentration to resin modified glass ionomer cement for 15 days was more effective in inhibiting the growth of S. mutans. Conclusion: The addition of propolis to adhesive materials provides an inhibitory effect on the growth of S. mutans, which may be effective in the world of preventive dentistry.", "keywords": [ "Propolis", "Resin Modified Glass Ionomer Cement", "Inhibition Zone", "Streptococcus mutans." ], "content": "Introduction\n\nFixed orthodontic treatment can be a risk factor for plaque accumulation1, which is significantly affected by the presence of bracket attachments and archwires2. The large number of areas for microbial colonization during orthodontic treatment can cause plaque accumulation, especially around the bracket and cervical edge of the band3. As many as 60% of fixed orthodontic patients also show poor oral health, which is shown by the high plaque index value during orthodontic treatment and the presence of white spot lesions in ~50% of orthodontic patients4,5. A study also reported that the incidence of fixed orthodontic patients with one new white spot lesion during treatment was 72.9% and incidence of cavity lesions was 2.3%6.\n\nThe presence of fixed orthodontic appliance in the oral cavity increases microbial population. Observational studies have shown that there is a positive relationship between the use of fixed orthodontic appliance and the number of bacteria, such as Streptococcus mutans, on plaques, which is known as bacteria in early or initial caries7–9. These bacteria have the ability to attach to all surface locations in the oral cavity including the surface of the bracket and the area adjacent to the bracket10. Efforts to protect areas that are vulnerable to bacterial colonization needs to be done, especially the area around the bracket.\n\nFixed orthodontic treatment develops fast along with the development of adhesive materials used to attach brackets to the tooth surface11. Brackets are attached to the teeth using acid etching or cemented using glass ionomer cement8. Resin modified glass ionomer cement is developed by adding hydrophilic resins such as hydroxydimethacrylate and BIS-GMA to conventional glass ionomer cement. This material is not only known to be attached to the surface of the tooth, but is also able to release fluoride, has better physical properties, shorter setting time, and is more effective against humidity12. At present, new research has been performed to develop a dental material with antibacterial activity, which will play an important role in preventing caries. Glass ionomer cement as bracket adhesive material can release fluoride but the desired antibacterial effect needs to be enhanced13.\n\nOver the past few decades, the use of natural products for pharmacological purposes has increased in the world14. Propolis is a sticky resin substance collected by honey bees from the sap of plants, leaves, and buds, which are mixed with the sap and saliva of bees in the nest. There are more than 180 chemical substances contained in propolis and are influenced by the type of bee, climate, plants and trees, and the time of collection. Bees use propolis to strengthen the nest wall and protect it from infection, and the human population use this product for many purposes15, for example propolis is known to provide protection against cariogenic bacteria and oral pathogens16. Hasan et al.17 stated that there are few studies about propolis activity from Indonesia.\n\nPropolis, as a natural product, can be used in a mixture of glass ionomer cement in order to increase antibacterial activity13,18. Megawati19 investigated the shear bond strength of metal brackets bonding to resin modified glass ionomer cement adhesive materials combined with 25% and 50% propolis. The addition of 50% propolis concentration had better shear bond strength and was able to withstand shear bond strength of 6–8 MPa according to the standard strength of clinically acceptable adhesive materials. Further research on the effect of the addition of 0%, 15%, and 25% Indonesian propolis for 0, 15 and 30 days on resin modified glass ionomer cement to the growth inhibition zone of S. mutans has never been done. The present study was an in vitro experimental study done in order to determine the antibacterial activity of resin modified glass ionomer cement combined with Indonesian propolis on S. mutans. The hypothesis was that adding propolis to this orthodontic bracket adhesive will increase the antibacterial properties of the adhesive.\n\n\nMethods\n\nEthical clearance for the study was obtained from Faculty of Dentistry, Universitas Gadjah Mada, Yogyakarta, Indonesia (No.001621/KKEP/FKG-UGM/EC/2018).\n\nPure propolis was produced by honeybees (Apis mellifera) in Indonesia (Figure 1). The propolis (970g) was purchased from Klinik Apitheraphy Kusuma (Moyudan, Sleman, Daerah Istimewa Yogyakarta). Propolis samples was chopped into small pieces and ground using a blender. Then, each 250 g sample of propolis was dissolved in 2500 mL of ethanol 80%, stirred at 800 rpm for 30 minutes using an electric stirrer (RW 20 digital; IKA, Germany) and left for 24 hours at room temperature. Rough particles were removed from the propolis extract using rough filter paper (58 cm × 58 cm) and the propolis was stirred once again for 30 minutes using an electric stirrer, left for 24 hours, and filtered. The filtrate was concentrated by a vacuum rotary evaporator. Next, the extract is poured in a porcelain cup and heated with a waterbath (70 °C) to produce propolis extract. Samples were kept in a dry and dark place until they were used19.\n\nConventional adhesive resin modified glass ionomer cement (Fuji Ortho LC, GC, Japan), which is made up of powder and liquid, was used in this study. Samples were prepared containing the conventional resin modified glass ionomer cement liquid and the two concentrations of propolis (15% and 25%) : (i) Resin modified glass ionomer cement with 0% propolis (PowderRMGIC:LiquidRMGIC ratio = 1:1) (n=15); (ii) resin modified glass ionomer cement with 15% propolis (PowderRMGIC: LiquidRMGIC:Propolis Extract ratio = 1:0.85:0.15) (n=15); (iii) resin modified glass ionomer cement with 25% propolis (PowderRMGIC: LiquidRMGIC:Propolis Extract ratio = 1:0.75:0.25) (n=15). Each group of samples was incubated for 0, 15, and 30 days (n=5/time duration). The adhesive materials were mixed according to the manufacturer instructions. After mixing the powder and liquid of each cement, samples were put into cylindrical molds (5 mm in diameter and 0.64 mm thickness), and the upper surface was flattened by pressing down and exposed by light curing unit (LY-B200, Liang Ya,China) for 10 seconds each surface (Figure 2)19,20.\n\nResin modified glass ionomer cement with (a) 0% propolis, (b) 15% propolis, and (c) 25% propolis.\n\nAgar disk diffusion test was performed at the Microbiology Laboratory, Faculty of Veterinary, Universitas Gadjah Mada, Indonesia. S. mutans ATCC 25175 type strain was used throughout the study. Bacterial strain from stock cultures was cultivated in Brain Heart Infusion broth at 37°C for 24 hours, corresponding to 108 CFU/mL using the McFarland scale. S. mutans was spread uniformly on the surface of Mueller Hinton Agar plates to produce a lawn. Adhesive samples were inserted in the plates. After a 24h incubation period in an incubator at 37°C, the plates were taken out of the incubator and the antibacterial activity was evaluated using a digital caliper to measure the diameter of halos of growth inhibition of the strain at three different points. The inhibitory zone was considered the distance (mm) from the outside margin of the samples to the initial point of the microbial growth (Figure 3). The mean was calculated for each sample and all measurements were performed by the same blinded operator21.\n\nLeft panel, measurement guidance; right panel, growth inhibition zone of S. mutans.\n\nData were analyzed using SPSS IBM for Windows (Version 22.0). The normality and homogeneity of variance in each group was confirmed before analyzing the data. Data of resin modified glass ionomer cement with 0%, 15%, and 25% propolis groups for 0, 15, and 30 days group were not normal and homogenous, so they were analyzed using Kruskal Wallis test and then Mann-Whitney test to determine the significance differences between groups. The significance level was set at 5%.\n\n\nResults\n\nPropolis, as a natural product, was combined with resin modified glass ionomer cement in order to assess antibacterial activity against S. mutans. The mean diameters of bacterial growth inhibited by different concentrations and duration of propolis combination in the adhesive are shown in Table 1. The growth inhibition zone in the present study was shown as a transparent or clear area around the adhesive materials.\n\nPercentages are propolis concentration in resin modified glass ionomer cement.\n\nTable 1 provides summarized data regarding the agar diffusion method and show the mean and standard deviation values of the diameter of growth inhibition zone for each sample in the groups against the S.mutans strain. Clear inhibition zones were shown showing that the propolis enhanced the antibacterial effect of the resin modified glass ionomer cement. Among the concentrations (0%, 15%, and 25% propolis), 0% propolis still showed inhibitory effect reflecting that the resin modified glass ionomer cement has its own antibacterial activity. The addition of 25% propolis revealed higher antibacterial activity than 15% and 0% propolis (Table 1). As can be seen in Table 1, the mean diameter of growth inhibition zone for 0 days was lower than 30 and 15 days. In general, the duration of propolis after 15 days resulted in a greater inhibition zones compared with 30 and 0 days.\n\nLooking at the data together (concentration and duration, it was observed that there was an interaction between concentration and duration of propolis to the growth inhibition zone against S.mutans (Table 2). The antibacterial activity of resin modified glass ionomer cement with 25% propolis for 15 days was the highest among all other concentrations for all tested days (p<0.05).\n\n*Statistically significant (p<0.05)\n\nI: Resin modified glass ionomer cement with 0% propolis; II: Resin modified glass ionomer cement with 15% propolis ; III: Resin modified glass ionomer cement with 25% propolis; A: 0 days; B: 15 days; C: 30 days\n\n\nDiscussion\n\nAn increase in the number of bacteria and plaques in the oral cavity of fixed orthodontic patients is one challenge for orthodontists. In the present study, a growth inhibition zone as a high concentration of propolis increased the antibacterial activity of the adhesive material to S.mutans, which was shown by an increase in the diameter of the growth inhibition zone. Resin modified glass ionomer cement with the addition of 25% propolis showed the largest diameter of the inhibition zone in all time periods, indicating the highest antibacterial activity compared to other treatment groups (15% and 0% propolis). Therefore, the addition of propolis to orthodontic adhesive materials as an antibacterial agent to inhibit the growth of Streptococcus mutans can be considered. This is in accordance with Asdar et al.22, who stated that propolis could inhibit the growth of S. mutans and it appeared in diameter changes of the inhibition zone. The greater the concentration of propolis, the greater the effect of inhibition produced.\n\nThe results agree with many researchers who have demonstrated the antibacterial activity of propolis. The mechanism of propolis against microorganisms is complex. Propolis works by inhibiting bacterial mobility and enzyme activity, as well as affecting the cytoplasmic membrane, which changed membrane permeability15. The functional and structural damage is suspected to occur due to components in propolis extract, such as flavonoids (quercetin, galangin, and pinocembrin), caffeic acid, benzoic acid, and cinnamic acid23,24. Koo et al.25 revealed that flavonoids, the largest component of propolis Apis mellifera, works by inhibiting glycosyltransferase activity. Extracellular polysaccharides, generally glucans, are produced by glucosyltransferase and play a role in the cariogenicity of dental biofilms26. Therefore, inhibition of glucosyltransferase interferes with cell metabolism through biochemical reactions and has the potential to prevent caries, especially in fixed orthodontic patients. Flavonoids interact with bacterial cell walls, forming complex compounds with extracellular proteins through hydrogen bonds so that they inhibit the activity of microorganisms, including bacterial mobility27. In addition, Pelczar and Chan28 showed that flavonoids denature and coagulate bacterial cell proteins so that cell damage cannot be repaired. Flavonoids could penetrate bacterial cell peptidoglycans so that the cell layer is not intact. The instability of cell walls cause the permeability of the cell and the control of the protein composition to be disrupted so that bacterial cells lose their shape and are lysed28. Pelczar and Chan28 also revealed that the higher the concentration of an antibacterial agent the stronger the antibacterial activity. An increased concentration of propolis added to the resin modified glass ionomer cement in the present study resulted in a darker color. The antibacterial ability of the resin modified glass ionomer cement increased with a higher propolis concentration. The results of the study were also in line with research conducted by Woo29, who concluded that the addition of antibacterial agents to glass ionomer cement, such as propolis, which had a darker color of propolis indicated more flavonoid as an active substance; therefore, the antibacterial activity increased with higher flavonoid content.\n\nThe present results showed that the duration of propolis treatment significantly affected the growth inhibition zone of S.mutans on resin modified glass ionomer cement. There was smaller diameter values of the growth inhibition zone on all types of adhesive materials with the addition of propolis for 30 days compared with 15 days which indicated lower antibacterial activity of the adhesive material. This is similar to research that examined the antibacterial effect of resin modified glass ionomer cement and concluded that there was an increase in the average diameter of inhibition zones over time, especially in the first week, namely days 1, 3 and 730.\n\nHigher antibacterial activity on day 15 than day 0 in the present study could be caused by several things, such as changes in pH and release of fluoride. An inhibition zone was suspected to be caused by the production of a low pH around the test material. The resin modified glass ionomer cement liquid component contained hydroxylethyl methacrylate (HEMA), which may have facilitated low pH and contributed to antibacterial properties31. Prasad and Maradia32 also added that the initial value of pH after mixing was also acid, where most bacterial growth would be suppressed then the pH value began to increase to a neutral level where it was not enough to inhibit bacterial growth. Kavita et al.33 mention that an increase in pH and decrease in release of fluoride ions explains the decrease in antibacterial activity.\n\nFluoride inhibits the acid production and glucans by S. mutans, which has been demonstrated by Wiegand et al.34, who showed that the release of fluoride could reduce demineralization, increase remineralization, and inhibit bacterial growth so that glass ionomer cement was cariostatic. Additionally, Featherstone35 demonstrated that fluoride worked by inhibiting bacterial metabolism through changes in hydroxyapatite on enamel to fluorapatite so that the enamel was more resistant to acid and increased remineralization. The high release of fluoride in resin modified glass ionomer cement ocurred because the acid base reaction was slowed down by the resin component, causing the ionized matrix to be less mature and capable of releasing more fluoride when compared to other materials, such as composite resins; greater pore size and porosity in resin modified glass ionomer cement; lower solubility and higher proportion of liquid powder with high viscosity36. Material with a resin content that is slightly like resin modified glass ionomer cement has a higher porosity, which facilitates the diffusion of fluoride37. This is also in line with research of Fucio et al.38 who found that resin modified glass ionomer cement changed in fluoride ion release over time. In that study, the release of fluoride at the beginning of the study occurred because the glass particles reacted with polyalkenoic acid, while continuous fluoride release could be caused by the ability of fluoride to diffuse through the cement pore. According to Toba et al.39, the hydrophilic property of HEMA from resin modified glass ionomer cement is required for the water absorption process and helps the diffusion of fluoride, which causes an increase in the release of fluoride ions.\n\nIn the present study, smaller diameter of the inhibition zone on day 30 than day 15 indicated that the antibacterial effect of the material decreased over time. The result of this study was in accordance with Matalon et al.40 who showed that high antibacterial potency of resin modified glass ionomer cement at the beginning of their study decreased for the next 3 weeks even though the antibacterial material was durable. Therefore, fluoride release of resin modified glass ionomer cement could decrease significantly with long-term use41. The antibacterial activity of resin modified glass ionomer cement in the present study was lower on the 30th day.\n\nStatistical analysis in the present study showed that there was an interaction between concentration and duration of propolis to the growth inhibition zone of S. mutans (p<0.05). This means that our hypothesis was accepted. The results of the study were in accordance with the study by Dastjerdie et al.5 where the antibacterial activity of adhesive materials to the growth of S. mutans depended on the type of cement and time.\n\nDiameter of the inhibition zone in the present study was smaller on day 30 compared with day 15 with the addition of propolis; however, this is classified as a strong response (11–20 mm) compared to the response of resin modified glass ionomer cement without addition of propolis, classified as a medium response (5–10 mm)42. A survey of 500 patients at a public health center in Jakarta and dental hospital at Universitas Indonesia conducted by Maringka and Herda43 showed that 90% respondents had experienced bracket detachment and around 60% of respondents experienced this event before their next appointment (three weeks after placement). Therefore, a strong response from the results of the day 30 treatment would be enough in the first 3 weeks. Although resin modified glass ionomer cement as an orthodontic adhesive material that releases fluoride has been used, the addition of propolis to orthodontic adhesive materials also provides an additional effect on the growth of S. mutans and was quite effective in this study.\n\n\nConclusion\n\nThe addition of propolis to adhesive materials gives an inhibitory effect on the growth of Streptococcus mutans. There was an interaction between concentration and duration of propolis and antibacterial effect against Streptococcus mutans.\n\n\nData availability\n\nFigshare: Growth Inhibition Zone Around Resin Modified Glass Ionomer Cement Bracket Adhesive, https://doi.org/10.6084/m9.figshare.10263275.v244.\n\nThis project contains the following underlying data:\n\n- Growth inhibition zone around resin modified glass ionomer cement bracket adhesive\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\nZachrisson S, Zachrisson BU: Gingival condition associated with orthodontic treatment. Angle Orthod. 1972; 42(1): 26–34. PubMed Abstract\n\nKloehn JS, Pfeifer JS: The effect of orthodontic treatment on the periodontium. Angle Orthod. 1974; 44(2): 127–34. PubMed Abstract\n\nMizrahi E: Enamel demineralization following orthodontic treatment. Am J Orthod. 1982; 82(1): 62–67. PubMed Abstract \| Publisher Full Text\n\nAtassi F, Awartani F: Oral hygiene status among orthodontic patients. J Contemp Dent Pract. 2010; 11(4): 1–10. PubMed Abstract\n\nVahid Dastjerdie E, Oskoui M, Sayanjali E, et al.: In-vitro Comparison of the Antimicrobial Properties of Glass Ionomer Cements with Zinc Phosphate Cements. Iran J Pharm Res 2012; 11(1): 77–82. PubMed Abstract \| Free Full Text\n\nRichter AE, Arruda AO, Peters MC, et al.: Incidence of caries lesions among patients treated with comprehensive orthodontics. Am J Orthod Dentofacial Orthop. 2011; 139(5): 657–64. PubMed Abstract \| Publisher Full Text\n\nMarsh PD, Martin MV, Williams D, et al.: Oral Microbiology. 5th ed. London: Elsevier. 2009; 14–26. Reference Source\n\nGill DS: Ortodonsia at a Glance. Jakarta: EGC. 2014; 35: 120–121.\n\nSturdevant CM, Roberson TM, Heymann HO, et al.: The art and science of operative dentistry. 3rd ed. St Louis: Mosby. 1995; 62.\n\nTanzer JM: Microbiology of dental caries. In: Contemporary oral microbiology and immunology. St Louis: Mosby. 1992; 377–422.\n\nGrandhi RK, Combe EC, Speidel TM: Shear bond strength of stainless steel orthodontic brackets with a moisture-insensitive primer. Am J Orthod Dentofacial Orthop. 2001; 119(3): 251–5. PubMed Abstract \| Publisher Full Text\n\nKumar M, Kumari S: Resin-Modified Glass Ionomer Cement and Its Use in Orthodontics-Concept Old is Gold: View Point. International Journal of Dental and Medical Specialty. 2016; 3(3): 10–4. Publisher Full Text\n\nHatunoğlu E, Oztürk F, Bilenler T, et al.: Antibacterial and mechanical properties of propolis added to glass ionomer cement. Angle Orthod. 2014; 84(2): 368–73. PubMed Abstract \| Publisher Full Text\n\nKumazawa S, Hamasaka T, Nakayama T: Antioxidant activity of propolis of various geographic origins. Food Chem. 2004; 84(3): 329–39. Publisher Full Text\n\nAhuja V, Ahuja A: Apitherapy- A sweet approach to dental diseases. part II: propolis. J Academy Adv Dental Research. 2011; 2(2): 1–8. Publisher Full Text\n\nPoeta P, Igrejas G, Goncalves A: Influence of oral hygiene in patients with fixed appliances in the oral carriage of antimicrobial-resistant Escherichia coli and Enterococcus isolates. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2009; 108(4): 557–64. PubMed Abstract \| Publisher Full Text\n\nHasan AEZ, Artika IM, Fatoni A, et al.: Anti-bacterial activity of propolis Trigona spp from Bukittinggi West Sumatera against Salmonella sp. Chem Prog. 2011; 4(2): 55–9. Reference Source\n\nTopcuoglu N, Ozan F, Ozyurt M, et al.: In vitro antibacterial effects of glass-ionomer cement containing ethanolic extract of propolis on Streptococcus mutans. Eur J Dent. 2012; 6(4): 428–33. PubMed Abstract \| Free Full Text\n\nMegawati V: Pengaruh penambahan propolis konsentrasi 25% dan 50% pada semen ionomer kaca modifikasi resin terhadap kekuatan geser pelekatan braket logam. [Thesis]. Yogyakarta: Universitas Gadjah Mada. 2017. Reference Source\n\nSodagar A, Akhoundi MSA, Bahador A, et al.: Effect of TiO2 nanoparticles incorporation on antibacterial properties and shear bond strength of dental composite used in Orthodontics. Dental Press J Orthod. 2017; 22(5): 67–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYusuf N: Pengaruh penambahan propolis Trigona sp pada bahan tumpatan glass ionomer cement terhadap pertumbuhan Streptococcus mutans (Penelitian in vitro). [Undergraduate Thesis]. Makasar: Universitas Hasanuddin; 2016. Reference Source\n\nAsdar, Hasanuddin, Asad S, et al.: Antibacterial activity test of South Sulawesi Propolis extract against Streptococcus mutans. Sch J Dent Sci. 2015; 2(2B): 195–8. Reference Source\n\nMarcucci MC: Propolis: chemical composition, biological properties and therapeutic activity. Apidologie. 1995; 26(2): 83–99. Publisher Full Text\n\nMirzoeva OK, Grishanin RN, Colder PC: Antimicrobial action of propolis and some of its components: the effects on growth, membrane potential and motility of bacteria. Microbiol Res. 1997; 152(3): 239–46. PubMed Abstract \| Publisher Full Text\n\nKoo H, Rosalen PL, Cury JA, et al.: Effects of compounds found in propolis on Streptococcus mutans growth and on glucosyltransferase activity. Antimicrob Agents Chemother. 2002; 46(5): 1302–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRen Z, Chen L, Li J, et al.: Inhibition of Streptococcus mutans polysaccharide synthesis by molecules targeting glycosyltransferase activity. J Oral Microbiol. 2016; 8(1): 31095. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIkeno K, Ikeno T, Miyazawa C: Effects of propolis on dental caries in rats. Caries Res. 1991; 25(5): 347–51. PubMed Abstract \| Publisher Full Text\n\nPelczar MJ, Chan ECS: Dasar-dasar mikrobiologi. Jakarta: Universitas Indonesia; 1988. Reference Source\n\nWoo KS: Use of Bee Venom and Propolis for Apitherapi in Korea. Philippines: Symposium and Technofora. 2004; 311–5. Reference Source\n\nBora TD, Tirali RE, Cehreli SB, et al.: The antibacterial and shear peel bond strength properties of different dental luting cements. Acta Scientific Dental Sciences. 2018; 2(3): 44–9. Reference Source\n\nVermeersch G, Leloup G, Delmée M, et al.: Antibacterial activity of glass-ionomer cements, compomers and resin composites: relationship between acidity and material setting phase. J Oral Rehabil. 2005; 32(5): 368–74. PubMed Abstract \| Publisher Full Text\n\nPrasad MP, Maradia MA: Antibacterial activity of conventional and modified glass ionomer cement against Streptococcus mutans. J App Biol Biotech. 2014; 2(3): 17–20. Reference Source\n\nKavita H, Thosar N, Baliga S, et al.: Antibacterial effects of hybrid tooth colored restorative materials against Streptococcus mutans: An in vitro analysis. J Conserv Dent. 2013; 16(4): 319–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWiegand A, Buchalla W, Attin T: Review on fluoride-releasing restorative materials--fluoride release and uptake characteristics, antibacterial activity and influence on caries formation. Dent Mater. 2007; 23(3): 343–62. PubMed Abstract \| Publisher Full Text\n\nFeatherstone JD: The science and practice of caries prevention. J Am Dent Assoc. 2000; 131(7): 887–99. PubMed Abstract \| Publisher Full Text\n\nDionysopoulos P, Kotsanos N, Pataridou A: Fluoride release and uptake by four new fluoride releasing restorative materials. J Oral Rehabil. 2003; 30(9): 866–72. PubMed Abstract \| Publisher Full Text\n\nXu X, Burgess JO: Compressive strength, fluoride release and recharge of fluoride-releasing materials. Biomaterials. 2003; 24(14): 2451–61. PubMed Abstract \| Publisher Full Text\n\nFucio SB, Paula AB, Sardi JC, et al.: Streptococcus Mutans Biofilm Influences on the Antimicrobial Properties of Glass Ionomer Cements. Braz Dent J. 2016; 27(6): 681–7. PubMed Abstract \| Publisher Full Text\n\nToba S, Pereira PNR, Nikaido T, et al.: Effect to topical application of fluoride gel on artificial secondary caries inhibition. Int Chinese J Dent. 2003; 3: 53–61. Reference Source\n\nMatalon S, Slutzky H, Weiss EI: Antibacterial properties of 4 orthodontic cements. Am J Orthod Dentofacial Orthop. 2005; 127(1): 56–63. PubMed Abstract \| Publisher Full Text\n\nBehrend B, Geurtsen W: Long-term effects of four extraction media on the fluoride release from four polyacid-modified composite resins (compomers) and one resin-modified glass-ionomer cement. J Biomed Mater Res. 2001; 58(6): 631–7. PubMed Abstract \| Publisher Full Text\n\nDavis WW, Stout TR: Disc plate method of microbiological antibiotic assay. I. Factors influencing variability and error. Appl Microbiol. 1971; 22(4): 659–65. PubMed Abstract \| Free Full Text\n\nMaringka G, Herda E: The duration of bracket detachment at public health center Jakarta and dental hospital Universitas Indonesia. J Int Dent Med Res. 2016; 9: 345–50. Reference Source\n\nSaputra SK, Farmasyanti CA: Growth Inhibition Zone Around Resin Modified Glass Ionomer Cement Bracket Adhesive. figshare. Thesis. 2019. http://www.doi.org/10.6084/m9.figshare.10263275.v2" }	[ { "id": "60534", "date": "23 Mar 2020", "name": "Ida Bagus Narmada", "expertise": [ "Reviewer Expertise Herbal in Orthodontics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, please allow me to congratulate the authors for attempting to undertake this study which I found quite interesting and useful as a reference for further study in the orthodontics material field. The manuscript itself is well-written and well-structured and I have to also commend the authors for this matter.\nHowever, I may require some clarifications on the following issues:\nThe paper is well organized and easy to follow.\n\nTo improve the readability, it is recommended that the text is checked by a native English speaking person as many of the sentences might be misunderstood. I suggest a revision of the grammar structures by an expert editor in revising manuscripts.\n\nAbstract:\nPlease add the p-value used for the Kruskal Wallis test (p<0.05 or p<0.01) - add homogeneity and normality test p-value used (p>0.05) in the abstract section.\n\nPlease add the exact p-value obtained after the statistic analysis (p=.....).\n\nPlease check the keywords according to the MESH NCBI data base and sort alphabetically.\n\nIntroduction:\nPlease add your hypothesis.\n\nPlease add a statement or sentence about whether there are any similar studies that have been done before or not? Or is your study the first study to investigate this topic?\n\nPlease add the critical or important issue within this study.\n\nPlease add the reason why only S. mutans was examined in this study.\n\nMaterial and Methods:\nIt would better if you add the detailed protocol or cite the protocol reference.\n\nIt would better if you add the confirmation of S. mutans colony or the detail of ATCC.\n\nIt would be better if you add more specific methods than agar disk diffusion test to examine the combination of propolis and GIC inhibit the S. mutans.\n\nIn this study, the authors performed the Kruskal wallis test. It would be better if the authors also described the Levene's and Shapiro-Wilk test.\n\nResults:\nIt would be better to understand and attract the reader if you present the data in the diagrams.\n\nPlease add the results of the Levene's and Shapiro-Wilk tests . Please indicate whether the difference is significant or not in your graph/diagrams as well, using some symbols (asterisk).\n\nDiscussion:\nDiscussion of the results is quite comprehensive. In analyzing the results, the authors also show citations from the previous study to support the explanation of these results.\n\nThe answer to the hypothesis of this study has been included at the beginning of the discussion section.\n\nPlease mention the limitation(s) of this study in the discussion section.\n\nReferences:\nThe supporting references are inadequate, please add the newest references that support this study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5384", "date": "03 Apr 2020", "name": "Stefani Kristanti Saputra", "role": "Author Response", "response": "Dear Prof Ida Bagus Narmada & Dr Alexander Patera Nugraha,Thank you for your kind assistance in reviewing our manuscript and providing us with valuable advices. Please allow us to comment as follows: Abstract:1. Please add the p-value used for the Kruskal Wallis test (p<0.05 or p<0.01) - add homogeneity and normality test p-value used (p>0.05) in the abstract section.2. Please add the exact p-value obtained after the statistic analysis (p=.....). The significance value of the normality and homogeneity test was (p> 0.05) while the significance value of the Kruskal Wallis test was (p <0.05).Datas in this study that were not normally distributed (p = 0.012) but homogeneous (p = 0.110) were analyzed by the Kruskal-Wallis test (p = 0.003) and then the Mann-Whitney test was performed to determine differences in significance between treatment groups. The significance value of the Mann-Whitney test was (p<0.05).3. Please check the keywords according to the MESH NCBI data base and sort alphabetically.Disk Diffusion Antimicrobial Tests. Glass Ionomer Cement. Propolis. Streptococcus mutans. Introduction:1. Please add your hypothesis.There is an relationship between concentration and duration of propolis to the growth inhibition zone of S.mutans. Adding propolis to orthodontic bracket adhesive will increase the antibacterial properties of the adhesive.2. Please add a statement or sentence about whether there are any similar studies that have been done before or not? Or is your study the first study to investigate this topic?Hatunoglu et al. (2014) has investigated that the addition of 25% and 50% Turkish propolis to glass ionomer cements increased antibacterial activity without modifying the mechanical properties of glass ionomer cements13 but Hatunoglu et al. (2014) have not conducted time-related research. Research conducted by Megawati (2017)19 shows that the addition of 25% concentration of propolis to resin modified glass ionomer cement does not affect shear strength19. Further research on the effect of adding propolis concentrations of 0%, 15%, and 25% for 0, 15, and 30 days in resin modified glass ionomer cements on Streptococcus mutans growth inhibition zones has never been done. Therefore, our research was the first study to investigate the relationship between concentration and duration of propolis to the growth inhibition zone of S.mutans.3. Please add the critical or important issue within this study.The presence of fixed appliances in the oral cavity of orthodontic patients increases the microbial population, such as Streptococcus mutans known as bacteria in early or initial caries. Glass ionomer cement is known to release fluoride but the desired antibacterial effect is still lacking.13 The antibacterial potential of propolis added to resin modified glass ionomer cement needs to be further investigated because orthodontic treatment takes place over a long period of time.4. Please add the reason why only S. mutans was examined in this study.S. mutans plays an important role in the etiology of caries. S. mutans is the first bacteria to form a colony and be the initiator of other bacteria to do the same thing.22 Material and Methods:1. It would better if you add the detailed protocol or cite the protocol reference.For the preparation of propolis extract, it refers to the research conducted by Megawati (2017)19.For the preparation of resin modified glass ionomer cement containing propolis, it refers to to the research conducted by Megawati (2017)19 and Sodagar et al. (2017)20. For the agar disk diffusion test, it refers to the research conducted by Yusuf (2016)21.2. It would better if you add the confirmation of S. mutans colony or the detail of ATCC.The Streptococcus mutans used in our study was pure culture from the isolate laboratory of the Microbiology Faculty of Veterinary Medicine UGM, Yogyakarta, Indonesia.3. It would be better if you add more specific methods than agar disk diffusion test to examine the combination of propolis and GIC inhibit the S. mutans.The diffusion method is most widely used to determine the sensitivity of bacteria to material because it is relatively simple and inexpensive. Unfortunately, it cannot determine the bactericidal and bacteriostatic properties of the drug. Hatunoglu (2014) has used a dilution method to determine the value of Minimum Inhibitory Concentration (MIC) of antibacterial material as the antibacterial capacity of glass ionomer cement with propolis13. This has not been included in this project but it will surely be considered for our future work.4. In this study, the authors performed the Kruskal wallis test. It would be better if the authors also described the Levene's and Shapiro-Wilk test. Saphiro-Wilk normality test is conducted to determine the distribution of data and the Levene homogeneity test is carried out to determine the homogeneity of data variance. Data that is not normally distributed or not homogeneous are analyzed by the Kruskal-Wallis test and then the Mann-Whitney test was performed to determine differences in significance between treatment groups. The significance value of the normality and homogeneity test was (p> 0.05) while the significance value of the Kruskal Wallis and Mann-Whitney test was (p <0.05). Results:1. It would be better to understand and attract the reader if you present the data in the diagrams. Please indicate whether the difference is significant or not in your graph/diagrams as well, using some symbols (asterisk).We will provide the data in the diagram with the new version of the article.2. Please add the results of the Levene's and Shapiro-Wilk testsThe results of the Shapiro-Wilk test was data in this study that were not normally distributed (p = 0.012<0.05) and the results of the Levene test was the datas were homogeneous (p = 0.110>0.05).Discussion:Please mention the limitation(s) of this study in the discussion section.Propolis is sufficient to be used as an antibacterial additive to resin modified glass ionomer cement, but it still needs further research aimed at the following:1. get a combination of physical properties, propolis concentration, shear strength, and tensile strength that meet the optimal standard of orthodontic adhesive; and2. increase stability and adequate propolis resistance. We thank you again for this valuable insight." } ] }, { "id": "61777", "date": "08 Apr 2020", "name": "Lidia Audrey Rocha Valadas", "expertise": [ "Reviewer Expertise Cariology", "Natural Products" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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:\nYou should justify the reason for studying just S.mutans, why not more species related to orthodontics fixed appliances?\n\nMaterials and methods:\nYou should cite the georeferencing of the extract and the chemical analysis performed to identify the main constituents.\n\nI don’t agree with the statement about 3 groups with different concentrations - I suggest 2 groups and one control. 0% is not a propolis concentration group.\n\nDiscussion:\nAdd the limitations of the study.\n\nPlease check reference 34, it is a review, in your discussion you cited it as a research article.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5423", "date": "15 Apr 2020", "name": "Stefani Kristanti Saputra", "role": "Author Response", "response": "Dear Dr Lidia Valadas,Thank you for your kind assistance in reviewing our manuscript and providing us with valuable advice for the manuscript and our future work. Please allow us to comment as follows:Introduction:You should justify the reason for studying just S.mutans, why not more species related to orthodontics fixed appliances?The presence of fixed appliances in the oral cavity of orthodontic patients increases the microbial population, such as Streptococcus mutans known as bacteria that plays an important role in the etiology of caries. S. mutans is the first bacteria to form a colony and be the initiator of other bacteria to do the same thing.22 Material and Methods:1. You should cite the georeferencing of the extract and the chemical analysis performed to identify the main constituents.The propolis was purchased from Klinik Apitheraphy Kusuma (Moyudan, Sleman, Daerah Istimewa Yogyakarta, Indonesia) and the production of the extract was done in LPPT Unit 1, Universitas Gadjah Mada, Yogyakarta, Indonesia. The pure propolis was produced by honeybees (Apis mellifera) and the main constituent of propolis Apis mellifera is flavonoid.2. I don’t agree with the statement about 3 groups with different concentrations - I suggest 2 groups and one control. 0% is not a propolis concentration group.We thank you for this valuable insight and it will be considered in our revision. Discussion: 1. Add the limitations of the study.Propolis is sufficient to be used as an antibacterial additive to resin modified glass ionomer cement, but it still needs further research aimed at the following:a. get a combination of physical properties, propolis concentration, shear strength, and tensile strength that meet the optimal standard of orthodontic adhesive; andb. increase stability and adequate propolis resistance.2. Please check reference 34, it is a review, in your discussion you cited it as a research article.Yes, it is a literature review. The purpose of the article was to review the fluoride release and recharge capabilities, and antibacterial properties, of fluoride-releasing dental restoratives, and discuss the current status concerning the prevention or inhibition of caries development and progression. We will revise and update the discussion section.We thank you again for the valuable insight." } ] } ]	1	https://f1000research.com/articles/8-2105
https://f1000research.com/articles/9-772/v1	27 Jul 20	{ "type": "Brief Report", "title": "What industries received COVID-19 closure orders? A cross-state comparison in the US", "authors": [ "Philip Jacobs", "Arvi P. Ohinmaa", "Arvi P. Ohinmaa" ], "abstract": "The United States federal government developed a COVID-19 blueprint for states to follow; it included the issuance by state/local governments of “stay at home” orders coupled with lists of essential services. Suppliers of these services would be exempt from closure so their workers could fulfill their essential functions. The blueprint was embraced by the states in a variety of ways. In this paper, we identify how business closure rules were enacted across the states for each of 15 types of services. The outcome measures were: “open” “open with restrictions” and “closure”. For six business types, most states permitted businesses to open. In four types, businesses were mainly closed. In three, they were allowed to open with restrictions. In the rest, there was a mixture of outcomes. In sum, the federal blueprint resulted in a regulatory patchwork as it spread throughout the country.", "keywords": [ "COVID-19", "Essential suppliers", "Economy" ], "content": "Introduction\n\nWhen the coronavirus (COVID-19) pandemic took hold in the U.S., the federal government developed a blueprint for the states to follow1. This plan included stay-at-home orders and a list of closure-exempted essential services, which was developed by the Cybersecurity and Infrastructure Security Agency (CSIA)2. In 42 states, governors issued state-wide stay-at-home mandates. Guided by the CSIA list, 28 states developed lists of non-essential services and used this list to mandate business, government, and nonprofit closures. In total 13 states developed their own “non-essential” lists along with their stay-at-home mandates. In six states (AR, IA, NE, ND, OK, WY), closures or reduced activities of non-essential services were ordered along with stay-at-home recommendations (but not mandates). And in two states (UT, SD) similar orders were made by regional governments3. A patchwork of rules regarding business closures emerged across the states4. Policy makers and researchers will be attempting to measure the effects of this pandemic on health, economic, and social outcomes for years to come, and an important component of these analyses will be an understanding of how policy responses regarding closure varied across the states. This study follows the designation of essential and non-essential industries in each state and summarizes them.\n\n\nMethods\n\nOur target variable was state governments’ designations of specific industries as essential, non-essential, or in-between. Essential industries were allowed to remain open; non-essential industries were required to close; in-between industries were allowed to open under restricted conditions.\n\nFirst, we obtained information on state closure policies from national newspaper reviews, the US Chamber of Commerce webpage, city newspaper articles, and state governor orders and supporting state documents (see Underlying data5: Appendix). Information was obtained using Google searches. Closures were recorded up to April 8, 2020 at which time all states had made closure announcements, and several were even considering reopening.\n\nSecond, we selected the industries of interest. We focused on those industries whose closure elicited discussion regarding closure orders; our sources were newspaper and broadcasting reports on essential/non-essential designations (see Underlying data5: Appendix for key references).\n\nThird, we obtained data on closure policies by state and industry. We used Google searches to identify state closure policies in regional and national newspaper and broadcasters’ web articles, on trade publications and websites of trade associations and on state orders. We primarily used these search terms: COVID-19, essential, mandate, closures and industry name/business line (e.g., florists) (for key references by industry see Underlying data5: Appendix).\n\nNon-essential services were classified into five groups, based on 6,7. These groups were: retail services, personal services (mostly one-on-one close contact, like nail salons), crowded areas (inside or outside), suppliers who provided personal services in crowded venues (e.g. restaurants and bars, schools, and child care centers), and upstream suppliers of non-essential services (e.g. plastic chip makers for casinos).\n\nAs the time the orders were issued varied by state, the governors’ proclaimed date (see Underlying data5) was used as the closure date. Generally, those governors who did so had acted by the end of the first week of April 2020. In a few states mentioned above closure policies were introduced by individual counties or cities, but a general state trend could be identified.\n\nClosure policies for each industry were divided into three groups – open (green in Figure 1), open but with restrictions (orange), or closed (red). The bars containing all three outcomes sum up to 50 (states) except in states where policies could not be classified.\n\n\nResults\n\nIn the retail business category 46 states deemed gun shops to be essential businesses. Closure orders were introduced but were being contested in the four other states. Greenhouses and garden centers were mostly considered as essential because of their relation to agriculture and food, although several states only allowed them to open under restricted conditions. Florists tried to be considered as part of this agricultural group, but in 33 of 42 states they were only allowed to remain open under restrictions; in these cases curbside pickups or home delivery were the main form of consumer contact. The upstream flower business, with its ephemeral production time was adversely affected as well.\n\nIn the personal services category, barber shops, hairdressers and nail salons were deemed to be non-essential in all states. After some controversy, all states deemed abortion clinics to provide essential services; however, in two states abortion clinics were required to operate with restrictions.\n\nIn the crowded venue category, state parks were allowed to be fully open in four states, and in 11 states attendance was restricted to daytime visitors. Golf courses remained open in 38 states, restricted in 4, and closed in 8. In four states, churches were allowed to remain open. In the remaining states they had to find other methods of communicating with parishioners.\n\nIn the category that combines personal services and crowding, gyms and health clubs were required to close in all states. In 38 states, hotels and motels were allowed to remain open, although there were restrictions, including limitations on meals and drinks served in common areas on the premises. Restaurants were closed to dining-in in 49 states, although in all states they were allowed to provide meals for pickup or delivery. Schools were closed in all states, but a few reopened under local school board authorities. Of the 45 states, where definitive information on childcare center policies could be obtained, complete closure was ordered in only one state. Centers remained open in 21 states. In the remaining 23 states where information could be obtained, the centers were allowed to remain open under restricted conditions: in ten of these states the number of children had to be less than 11, and in the other 13 states the centers could only take in children whose parents worked in essential industries.\n\nThe fifth category involves upstream producers. This category includes suppliers to non-essential service providers (e.g., flower growers for florists or producers of poker chips for casinos). CISA listed construction as an essential industry and many states have included light construction in the list of essential services. However, three states (MI, NY, NJ) adopted a stricter interpretation of essential construction; they excluded construction related to non-essential services. Also 40 states have included all construction as essential. In seven states the interpretation of “essential” for construction was more limited.\n\n\nDiscussion\n\nClosure policies have had a considerable impact on unemployment8,9. In a few industries (florists, state parks, golf courses) state government policies varied widely, but in most cases the results show general agreement between states. Compromises between complete closures and unrestricted openings occurred in a few industries (curbside delivery for restaurants and florists), although direct as well as upstream economic damage still occurred10–12. Even in cases where there was widespread agreement about what is “essential” (abortion, gun shops, churches), there were controversies and court challenges13–15.\n\nIn a few industries, the sum of state orders was less than 50. In these states, industry sources and/or governments themselves could not determine whether an order pertained to a specific group (e.g., florists as an agricultural or retail industry)16, and in some cases there was disagreement between governments within states, as when regional governments issued their own orders in addition to state-wide orders17 or instead of them18,19.\n\nThis paper identified state closure policies. In many states and industries, even when there were no closure orders, retail and service business still closed20. Thus the state policies do not fully reflect all business closures.\n\nClosure of non-essential industries was a key state and local policy in the U.S.\n\nClosure of an industry has important implications for owner investment, employment and consumption\n\nThere was agreement among the states for most industries, but strong disagreement for several, including golf courses and childcare centers\n\n\nData availability\n\nUniversity of Alberta Dataverse: COVID19 state closures by industry, https://doi.org/10.7939/DVN/YEDHP85.\n\nThis project contains the following underlying data:\n\n- A list of data sources (Appendix)\n\n- A spreadsheet of the status of the non-essential business for all 50 states as of April 8, 2020\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nWhite House: The president’s coronavirus guidelines for America.: 30 days to slow the spread. 2020; Accessed on May 10, 2020. Reference Source\n\nCISA: IDENTIFYING CRITICAL INFRASTRUCTURE DURING COVID-19. Original release date: March 18, 2020 \| Last revised: April 17, 2020. Retrieved on April 24, 2020. Reference Source\n\nMultiState Associates: Stay-at-Home Orders: What is Considered an “Essential Business”? Covid-19 state policy tracker. Alexandria, VA Multistate. 2020. Accessed on April 24, 2020. Reference Source\n\nAndrew S: What constitutes ‘essential businesses’? States seem to have varying standards. CNN. 2020. Reference Source\n\nJacobs P: COVID19 STATE CLOSURES BY INDUSTRY. UAL Dataverse, DRAFT VERSION 2020. http://www.doi.org/10.7939/DVN/YEDHP8\n\nColey SD, Woelfel TE: Guidance for Businesses Resolving Multiple “Shelter in Place” Orders and Differing Definitions of “Essential Business”. National Law Review. 2020. Reference Source\n\nWomble, Bond and Dickinson: Shelter In Place Orders: Are You an “Essential Business?” Acessed on May 15, 2020. Reference Source\n\nGascon C, Amburgay A: COVID-19, School Closings and Labor Market Impacts. On the Economy Blog. Federal Reserve Bank of St. Louis, 2020. Reference Source\n\nRobertson J: Unpaid Absence from Work Because of COVID-19. Macroblog. Federal Reserve Bank of Atlanta. 2020. Reference Source\n\nFaux Z, Herbling D, Munsterman R: The Crash of the $8.5 Billion Global Flower Trade. Bloomberg Businessweek. 2020. Accessed on July 12, 2020. Reference Source\n\nSchneider L: Dairy farmers dumping milk amid COVID-19: Pandemic's impact on the dairy industry. ABC News. 2020. Accessed on July 12, 2020. Reference Source\n\nJohansson R: Will COVID-19 Threaten Availability and Affordability of our Food? USDA Media Blog. 2020. Accessed on July 12, 2020. Reference Source\n\nPazanowski A: Louisiana Covid-19 order challenged by abortion providers. Bloomberg Law. 2020. Accessed on July 12, 2020. Reference Source\n\nLevin D: Coronavirus and firearms: are gun shops essential businesses? New York Times. 2020. Accessed on July 12, 2020. Reference Source\n\nHiggins T: Churches allowed to stay open in states where millions are particularly vulnerable to coronavirus. CNBC. 2020. Accessed on July 12, 2020. Reference Source\n\nWulff R: Coronavirus shutdown: are florists essential businesses? CBS13 Sacremento. 2020. Accessed on July 12, 2020. Reference Source\n\nMock B: These States Are Sowing Confusion About Cities’ Power to Fight Covid-19. Bloomberg City: Lab. 2020. Accessed on July 12, 2020. Reference Source\n\nSemerad T: How Utah’s coronavirus restrictions differ by county. Salt Lake City Tribune. 2020. Accessed on July 12, 2020. Reference Source\n\nGroves S: South Dakota Cities, Counties Take Action on COVID-19. Associated Press to U.S. News & World Report. 2020. Accessed on July 12, 2020. Reference Source\n\nRetail Dive Staff: Tracking retail’s response to the coronavirus. RetRetail Dive, 2020. Accessed on July 12, 2020. Reference Source" }	[ { "id": "68022", "date": "13 Aug 2020", "name": "Clara Dismuke-Greer", "expertise": [ "Reviewer Expertise I am a health economist with the US Department of Veterans Affairs" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 extremely important during this time of COVID and state and local policies on closures. While the figure is very nice and informative, I would very much like to see the underlying data provided in a table. This would help also with citations for future 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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5825", "date": "18 Aug 2020", "name": "Philip Jacobs", "role": "Author Response", "response": "We appreciate the reviewer's comments. The raw data by state is available in an Appendix to this article, published on Dataverse. The data can be obtained from this F1000 report, by going to the bottom of the page and downloading it from the Appendix called SERVICESBYSTATEXLM. It takes a bit of searching (I had to hunt for it myself) but it is there. Incidentally, all articles on F1000 do have data attached. It's a requirement for publication.Thanks again.Phil" } ] }, { "id": "68020", "date": "17 Aug 2020", "name": "Ceri J. Phillips", "expertise": [ "Reviewer Expertise Health economics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nA brief, descriptive analysis of closure policies across the US, in relation to designation of industries as essential, non-essential and those categorised as in-between.\nThe report provides a useful baseline of policies with which to subsequently assess the impact over time of the Covid-19 pandemic - in health, economic and social terms.\n\nIs the work 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/9-772
https://f1000research.com/articles/9-769/v1	24 Jul 20	{ "type": "Research Article", "title": "Cardiorespiratory physiological perturbations after acute smoke-induced lung injury and during extracorporeal membrane oxygenation support in sheep", "authors": [ "Saul Chemonges" ], "abstract": "Background: Numerous successful therapies developed for human medicine involve animal experimentation. Animal studies that are focused solely on translational potential, may not sufficiently document unexpected outcomes. Considerable amounts of data from such studies could be used to advance veterinary science. For example, sheep are increasingly being used as models of intensive care and therefore, data arising from such models must be published. In this study, the hypothesis is that there is little information describing cardiorespiratory physiological data from sheep models of intensive care and the author aimed to analyse such data to provide biological information that is currently not available for sheep that received extracorporeal life support (ECLS) following acute smoke-induced lung injury. Methods: Nineteen mechanically ventilated adult ewes undergoing intensive care during evaluation of a form of ECLS (treatment) for acute lung injury were used to collate clinical observations. Eight sheep were injured by acute smoke inhalation prior to treatment (injured/treated), while another eight were not injured but treated (uninjured/treated). Two sheep were injured but not treated (injured/untreated), while one received room air instead of smoke as the injury and was not treated (placebo/untreated). The data were then analysed for 11 physiological categories and compared between the two treated groups. Results: Compared with the baseline, treatment contributed to and exacerbated the deterioration of pulmonary pathology by reducing lung compliance and the arterial oxygen partial pressure to fractional inspired oxygen (PaO2/FiO2) ratio. The oxygen extraction index changes mirrored those of the PaO2/FiO2 ratio. Decreasing coronary perfusion pressure predicted the severity of cardiopulmonary injury. Conclusions: These novel observations could help in understanding similar pathology such as that which occurs in animal victims of smoke inhalation from house or bush fires, aspiration pneumonia secondary to tick paralysis and in the management of the severe coronavirus disease 2019 (COVID-19) in humans.", "keywords": [ "Sheep", "critical care", "smoke-induced acute smoke inhalation injury", "extra-corporeal life support", "lung compliance", "PaO2/FiO2 ratio" ], "content": "Introduction\n\nDuring multifaceted experiments involving intensive care in large animal models in translational research, information related to animal monitoring is often collected with varying accuracy, scope and end-user applications. Data collection can be manual, electronic, or both1–3. Manually input data can include subjectively scored end points, like the plane of anaesthesia, and objective data such as heart rate or breaths per minute. Depending on the goals of the study, certain information may be used to validate or test novel therapies, or to understand and refine existing treatments. In certain cases, experimental information may be collected for scientific curiosity or for ‘classified’ use, and outcomes may never be publicly available, particularly if the results are negative.\n\nThe source of data for this study was from a sheep model2–5 in which sheep were treated for acute smoke-induced acute lung injury using veno-venous (VV) extracorporeal membrane oxygenation (ECMO)2, a form of extracorporeal life support (ECLS) developed to complement the treatment of acute lung injury in humans6–8. During this type of ECLS, venous blood is carried from the patient to a gas exchange device where the blood is enriched with oxygen, has carbon dioxide is removed, and oxygenated blood is returned to the patient’s circulation in the right atrium. This method can be used for treatment, as respiratory support during lung transplantation, and in critically ill patients with potentially reversible respiratory failure8,9. The multiple advanced cardiovascular3, respiratory, patient point-of-care procedures and instrumentation associated with ECLS even in animal experimentation is highly data- and equipment-intensive. This platform is useful for developing research and methodological skills for in vivo animal instrumentation and for the processing of large, real-time clinical data sets from multifaceted animal studies that can be applied to similar intensive care scenarios. An opportunity to develop these skills arose within a source study conducted at Queensland University of Technology and The University of Queensland, which was an ongoing publicly funded animal experimentation study. While the objectives of the primary study had a separate focus, there were considerable amounts of redundant raw data with potential use in veterinary science and other disciplines, once processed.\n\nAlthough burn and smoke inhalation ECLS models in sheep have been around for many years, most studies have only focused on their translational potential for applications in human medicine and not for veterinary science applications or further refinement of the model. For example, it has been documented that prolonged exposure to smoke exacerbates lung injury after evaluating the manner various types of exposure to smoke chemicals cause injury10. Another sheep ECMO study investigated the pathophysiology of circulating leukocytes, oxygen free-radical activity, thromboxane release and respiration11. In the preceding study, animals treated with smoke injury followed by ECMO had significantly increased circulating thromboxane B2 levels and oxygen free-radical activity compared with controls and animals treated with smoke and mechanical ventilation but details of haemodynamics were not presented.\n\nIn the present study, the author hypothesised that there is little information describing physiological data from multifaceted sheep models of intensive care and the author aimed to analyse such data to provide cardiorespiratory biological information that is not currently available in sufficient detail for sheep that receive ECLS following acute smoke-induced acute lung injury. The overall goal was to provide useful information relevant to the sheep model itself as well as to those interested in broad animal experimentation and veterinary medicine in general. The specific objective was to utilise the raw data from the sheep ECMO model study and analyse that data to provide biological information that is not currently available for sheep that receive ECLS following acute smoke-induced lung injury to further understand the physiology of cardiorespiratory support.\n\n\nMaterials and methods\n\nAnimals were obtained and treated in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes12, with strict adherence to published and currently acceptable guidelines on using experimental animals and reporting findings13,14. All studies were registered with institutional animal welfare and ethics departments; moreover, the Queensland University of Technology Animal Ethics Approval No. 110000053 was obtained and it was ratified by The University of Queensland.\n\nBatches of approximately 2-year old healthy adult merino ewes (Ovis aries) were obtained and carted to Brisbane from the Australian Commonwealth Scientific and Industrial Research Organisation (CSIRO) breeding facility in Armidale, New South Wales. The sheep were agisted as a flock in an open pasture farm to be used as part of, and a continuation of previously described studies15. The farm had improved pastures and the sheep had natural shade from trees with free access to water before being transferred within two weeks of experiments to a purpose-built animal experimental laboratory at QUT-MERF1,15. The sheep were handled as per standard humane operating procedures that have been described in detail elsewhere1. The experiments were conducted at the Biological Research Facility (BRF) housed within the Medical Engineering Research Facility of Queensland University of Technology (QUT-MERF) in Brisbane. Animal selection and experimental procedures for this, and also other multifaceted studies that used similar methods have previously been described15–17. Briefly, the sheep were fed proprietary sheep pellets, lucerne and had free access to water. Shelter was provided in built concrete-floored sheds in which the sheep had free access. Shade was also provided by large trees in the paddocks and the sheep interacted freely with each other. Animals were fasted for approximately 24 h with free access to drinking water until two hours before the commencement of the experimental procedure at 8:00 – 9:00 a.m. on the day of the experiment. Local guidelines mandated the presence of a team comprising of a veterinarian and other trained personnel to monitor the sheep at all times for the duration of the experiment for animal welfare purposes and for the mitigating staff fatigue. If at any time the animal became physiologically distressed to such an extent that it could not be managed or reversed, mechanisms were in place to have the sheep be immediately euthanased and documented accordingly1.\n\nA sample size calculation was performed for the entire original sheep ECMO project which comprised 72 sheep (9 groups of 8 sheep each), as previously reported1. There was a subsequent addition of two control groups (two groups of eight sheep each), bringing the total number of sheep to 88. The other experimental groups were out of scope, as they investigated other aspects of ECLS such as blood transfusion studies, therefore, this study focuses on the cardiorespiratory physiology (4 groups) only. In this study, 19 sheep were pseudo-randomised into four experimental groups (Table 1). The experimental groups were classified based on the following two aspects: the duration of treatment (24 hours of ECMO only—E24H); treatment after smoke inhalation (injury) (24 hours of ECMO after smoke inhalation—SE24H). Two additional groups included one group that received smoke inhalation injury but no treatment (24 hours of monitoring only after smoke inhalation and no ECMO; SC24H), and another group that inhaled room air only as the injury (placebo) and no treatment (24 hours of monitoring, no smoke inhalation and no ECMO; C24H). Robust data was acquired from 16 sheep (E24H and SE24H) and included fully in the study; however, data of three sheep from groups SC24H and C24H was considered only as early observational data or case reports – within the timeline required to satisfy the requirements of a research degree at The University of Queensland back then. A systematic approach was developed for processing the data. (All raw and processed data are available as Underlying data and can be downloaded at http://www.doi.org/10.5061/dryad.3r2280gd518)\n\nTable 1 legend: BSA = Body surface area; E24H = uninjured sheep treated with extracorporeal life support (ECLS) for 24 hours (uninjured/treated); SE24H = sheep with acute smoke-induced lung injury treated with ECLS for 24 hours (injured/treated); SC24H = sheep with acute smoke-induced lung injury monitored for 24 hours without ECLS (injured/untreated); C24H = sheep subjected to room air injury as a control for smoke and monitored for 24 hours without ECLS (placebo/untreated).\n\nCritical care of animals, VV ECMO setup and physiological data acquisition. The details of animal selection, care, and pre-anaesthetic processes; anaesthesia technique; airway access and ventilation; instrumentation for VV ECMO; haemodynamic monitoring; respiratory monitoring; temperature, fluids, vasoactive drug administration, and electrolyte management; blood collection; physiological data acquisition; and the technique for euthanasia of the sheep after the experiments have previously been described in a detailed protocol1. In brief, the sheep was restrained in a sling cage and the ventral neck region was aseptically prepared to enable intravenous access. For VV ECMO implementation, venous blood was accessed from the right jugular vein of the animal and then oxygenated and returned to the right atrium of the heart after it was made to pass through an oxygenator. For the combined purpose of blood sampling, administration of medications, and fluid administration, a multi-lumen central venous catheter was inserted into the left jugular vein of the animal under local anaesthesia. The left jugular vein was also cannulated with an 8G sheath for the insertion of a pulmonary artery catheter for haemodynamic monitoring. In addition, an 11G sheath catheter was then inserted proximally into the left jugular vein for intra-cardiac echocardiography catheter insertion. The right jugular vein was cannulated both proximally and distally with single lumen central lines to aid insertion of return and access ECMO cannulas, respectively. All animals were intubated and received mechanical ventilation as previously described1. Briefly, the initial ventilator tidal volume was set to approximately 10 mL/kg with a respiratory rate of 15 breaths/min, positive end expiratory pressure (PEEP) of 5 cm H2O, and an initial FiO2 (fraction of inspired oxygen) of 1.0. These settings were then titrated based on arterial blood gas results. A low tidal volume—high PEEP strategy was used to minimise ventilator-induced lung injury.\n\nIn order to obtain high-quality cardiorespiratory monitoring data, the instrumentation of the sheep was undertaken to acquire and derive the following physiological parameters using established standard methods at defined timepoints: core body temperature (T), pO2, SpO2, alveolar–arterial oxygen gradient P(A-a)O2, PaO2/FiO2 ratio, end-tidal carbon dioxide concentration (etCO2), heart rate (HR), arterial blood pressure (BP) (systolic and diastolic), mean arterial BP (MAP), pulmonary artery pressure (PA) (systolic and diastolic), mean pulmonary artery pressure (MPAP), central venous pressure (CVP), pulmonary artery occlusion pressure (PAOP), mixed venous oxygen saturation (SvO2), stroke volume (SV), continuous cardiac output (CCO), cardiac index (CI), systemic vascular resistance (SV), systemic vascular resistance index (SVRI), pulmonary vascular resistance index (PVRI), left ventricular stroke work index (LVSWI), right ventricular stroke work index (RVSWI), coronary perfusion pressure (CPP), arterial oxygen content (CaO2), and oxygen delivery index (O2EI). (These methods are detailed in data files, available as Underlying data, which can be downloaded at http://www.doi.org/10.5061/dryad.3r2280gd518).\n\nSmoke inhalation injury. In the original study of the ECMO model4, sheep inhaled standardised cotton smoke generated by a device that combusts material in an oxygen-deficient environment as previously described19. In brief, 8 g of cotton towelling was combusted in a chamber with transparent walls and 400 ml tidal volume. One tidal volume breath (approximately 10–12 ml/kg) of the smoke was delivered to the sheep via plastic tubing that was 1 m long connected to a tracheostomy tube. A fixed number (12) of breaths were given with each load of cotton over a period of approximately one minute. Serial arterial blood gas samples were taken to assess the effect of smoke inhalation, beginning at a predetermined time point after the smoke breath cycles.\n\nRaw data were obtained from critical care monitoring of sheep undergoing treatment for acute smoke-induced lung injury that involved several separate previous projects. Data analysed were collected prior to 23 August 2013 and were kindly obtained from two of the scientists (Diab, S. and Dunster, K.R., who developed the base model – see reference 4), as part of a research higher-degree project of the author at The University of Queensland20,21. All data files were stored in Microsoft® Excel 97–2003 (Microsoft Corporation, Redmond, WA, USA) format and were grouped per sheep and date of the experiment. Data comprised separate files of real-time physiological data recorded in the hard drives of the monitoring devices (electronically acquired data) and parameters manually recorded by those monitoring the sheep under anaesthesia (manually acquired data)—which included data from the electronic monitoring equipment—as back-up if the electronic monitors malfunctioned.\n\nA clone of the master manual data entry Excel spreadsheet was created by excluding the formatting and formulas. Several members of the sheep ECMO research team repeatedly inspected the data for errors to in order to ensure that all columns, rows, time points, and data points had been copied correctly, including number formats. Redundant columns were omitted from the spreadsheet and data were curated and aligned to predetermined experimental time points. While maintaining the same experimental time point headers on the spreadsheet, data were grouped into the following categories: ventilator settings, blood pressure and haemodynamics, fluids and urine output, arterial blood gas values, activated clotting time, anaesthetics, anticoagulants, and ECLS circuit observations.\n\nElectronically acquired physiologic monitoring raw data were inspected for completeness. The data comprised 36 time points: ECLS pump time (min); time of day (h); electrocardiograph (heart rate); arterial blood pressure (mean, systolic, diastolic, heart rate); central venous pressure (mean); pulmonary artery pressure (mean, systolic, diastolic); oxygenator pressure (pre- and post-); capnography (etCO2, respiratory rate); pulse oximetry (SpO2, heart rate); ECLS pump (flow rate, speed); ventilator (mode, frequency, oxygen, pressure control, inspiratory volume, expiratory volume, expiratory minute volume, pressure maximum, mean pressure, positive end-expiratory pressure, plateau pressure, inspiratory resistance, expiratory pressure, pulmonary compliance, inspiratory flow); mixed venous oxygen saturation (SvO2); and continuous cardiac output (CCO). A baseline time point was established after instrumentation and an injury time point corresponding to the smoke inhalation time point was determined thereafter. It is important to note that there may or may not have been any data at any given point in time. The electronically acquired physiological monitoring data were inspected for errors and cleaned to provide data for downstream analysis.\n\nManually acquired observations at specifically designated timepoints were recorded and extracted as detailed in the Underlying data, filed at http://www.doi.org/10.5061/dryad.3r2280gd518. These timepoints were at baseline (soon after instrumentation of the sheep), smoke injury, 5 min post smoke injury, 1 hour post-smoke injury. This was then followed by ECMO treatment, which was recorded in the following manner: 0, 0.25, 1, 1.5, 2, 4, 6, 6.5, 7, 8, 10, 12, 14, 16, 18, 20, 22 and 24 hours of ECMO.\n\nThereafter, data were subjected to further integrity checks. An important step was to make a plot of data versus time together with descriptive statistics for all data points in the grouped data. After artefact elimination and integrity checks, data for individual sheep were assigned to six categories: activated clotting time; anaesthetics + inotropes and anticoagulants; arterial blood gas values; blood pressure + ventilation and haemodynamic data; calculated respiratory + haemodynamic variables; and fluids and urine production. Using specially written macros, data were extracted from each experiment and grouped by parameters corresponding to experimental time points. All sheep treatment data were then filed according to parameter.\n\nData from the 19 sheep from groups E24H, SE24H, C24H and SC24H were processed further. Data integrity checks were performed again and repeated by several sheep ECMO research team members. The treatment timeline comprised 22 time points for all experiments in which sheep received acute smoke-induced lung injury (SE24H). A trend plot and descriptive statistics panel in Excel were used for data quality control processes for suitability for downstream data analysis and end-user applications.\n\nIn order to meet the specific objective of the study, data from the groups, uninjured/treated and injured/treated groups were analysed after testing for normality using D’Agostino–Pearson omnibus normality test. The means, medians and standard deviations of the weights of the sheep, where applicable, were tabulated and graphically compared. The physiological parameters of the groups were charted and compared with each other using one-way analysis of variance (ANOVA) where appropriate, and significance was reported based on Brown-Forsythe test. Further, parameters between groups were compared using a paired two-tailed t-test. All p-values were two-sided and p < 0.05 was considered statistically significant. All statistical calculations were performed using GraphPad PRISM 6 software (GraphPad Software, La Jolla, CA, USA).\n\nAn earlier version of this article can be found on bioRxiv (DOI: https://doi.org/10.1101/058511).\n\n\nResults\n\nThe biodata of the sheep that were used in the current analysis are presented in the Methods section (see Table 1). The weights of the uninjured/treated sheep, unlike the injured/treated group, did not pass the D’Agostino–Pearson omnibus normality test; however, there was no significant difference in the weights of the sheep between the groups (Figure 1).\n\nA decrease in pulmonary compliance was found in all of the sheep during the course of the experiments, with the injured/treated (SE24H) animals having the most severe and drastic decrease followed by the uninjured/treated (E24H), injured/untreated (SC24), and placebo/untreated (C24) sheep in that order (Figure 2). There was a significant difference (p = 0.0013) in pulmonary compliance between uninjured/treated and injured/treated groups. The injured/treated sheep had consistently lower SpO2 compared with the other groups, but there was no significant difference in SpO2 readings between the groups (Figure 3). Further, there was an initial increase in etCO2 followed by a rapid decrease that reduced 15 minutes after the treatment was began. The etCO2 of the injured sheep continued to trend downward and plateaued in the uninjured groups (Figure 4). There was a significant difference (p = 0.0147) in the etCO2 between the uninjured/treated and injured/treated groups.\n\nDotted lines represent error bar margins.\n\nError bars are one-sided for clarity.\n\nBlood pH varied between the groups (Figure 5). The placebo/untreated sheep had the highest pH while the injured/treated group had the lowest. There was a significant difference in pH between the uninjured/treated and injured/treated groups (p = 0.0343). The pCO2 in all but the uninjured/treated sheep increased initially before plummeting sharply, thereby forming a shallow trough corresponding to 1 hour after the treatment, followed by a slight increase before stabilising in all sheep (Figure 6). There was a gradual decrease in pO2 in the treated groups of sheep from baseline before decreasing dramatically at the start of treatment with the injured sheep having the most profound decrease (Figure 7). However, there was no significant difference in pO2 between the uninjured/treated and injured/treated groups.\n\nThe dots represent hourly time-points.\n\nError bars have been omitted for clarity.\n\nError bars have been omitted for clarity.\n\nThe concentration of haemoglobin [Hb] was found to decrease slightly from baseline before gradually increasing in the injured sheep and remained relatively constant over time in the uninjured sheep. There was a significant difference in [Hb] between the uninjured/treated and injured/treated (p = 0.0131) groups (Figure 8). The fraction of oxyhaemoglobin (FO2Hb) decreased sharply with the lowest reading at five minutes post-injury before returning to near baseline levels within 1 hour of the treatment (Figure 9). The injured/treated sheep had a considerably deeper trough in FO2Hb level and there was a significant difference (p = 0.046) between troughs. There was no change in FO2Hb for the uninjured sheep. Further, the fraction of carboxyhaemoglobin (FCOHb) increased sharply from baseline, peaking at approximately five minutes post-injury and decreased sharply thereafter to the beginning of treatment before gradually returning to near-baseline levels at approximately 6 hours after the treatment was begun in the injured sheep (Figure 10). The injured/treated sheep had a higher peak FCOHb than the injured/untreated sheep, although the difference was not significant. There was no change in FCOHb for the uninjured sheep. The fraction of methaemoglobin (MetHb) increased gradually from baseline, peaking at approximately five minutes post-injury and then gradually decreased when the treatment was begun (Figure 11). This was followed by a gradual return to near-baseline levels at approximately 6 hours after the treatment was begun in the injured/treated sheep. There was no change in MetHb for the uninjured sheep. There was an initial subtle decrease in calculated haematocrit (Hct) before a steady increase in the injured sheep and relatively flat slopes for the uninjured sheep (Figure 12).\n\nError bars are one-sided for clarity.\n\nThe blood sodium concentration [Na+] was relatively stable and there were no significant differences between groups (Figure 13). There was an initial decrease in the blood calcium [Ca2+] level, with the lowest point at approximately 1 hour after the treatment was begun, before it levelled out thereafter in all groups (Figure 14). Further, there was a significant difference in [Ca2+] between the uninjured/treated and the injured/treated groups (p = 0.0001). The placebo/untreated and injured/treated groups maintained the highest and lowest levels of [Ca2+], respectively, throughout the experiments. Blood chloride [Cl-] levels remained stable compared with baseline levels during the initial stages and then increased gradually thereafter (Figure 15). The blood potassium concentration [K+] initially decreased as compared with baseline levels, reaching a minimum concentration 1 hour after the treatment was begun and then gradually increased with a peak at approximately 12 hours after treatment was begun in all experimental groups (Figure 16). Although the injured/untreated and injured/treated sheep had higher [K+] than the uninjured sheep, the differences were not significant. Overall, the anion gap decreased gradually, achieving a relatively gentle slope at approximately 6 hours after the treatment was begun and did not change significantly, thereafter (Figure 17). There was a gradual decrease in anion gap from baseline in the course of the experiments and there was no significant difference in anion gap between the uninjured/treated and injured/treated groups.\n\nError bars have been omitted for clarity.\n\nError bars have been omitted for clarity.\n\nAlthough there was an increase in blood glucose level [Glu] for the injured/treated sheep after 6 hours of treatment, the change was not significant. There was an initial decrease in lactate levels [Lac] 6 hours after the treatment was begun, followed by a gradual increase for the injured sheep, particularly for the injured/treated group. There was no significant difference in [Lac] between the treated groups.\n\nThere was an increase in the blood base levels [Base (ecf)] that peaked 1 hour post-treatment, followed by a gradual decrease in the untreated group. [Base (ecf)] in the treated groups remained at baseline levels to 1 hour after the treatment begun, before decreasing markedly in the injured/treated sheep (Figure 20). There was a significant difference (p = 0.0257) in base (ecf) between the uninjured/treated and injured/treated groups. Further, blood bicarbonate concentrations [HCO3-] increased initially in the untreated groups before decreasing gradually; however, levels remained higher compared with the treated sheep (Figure 21).\n\nThere was a gradual decrease in heart rate (HR) of the sheep during the course of the experiments, with the placebo/untreated groups maintaining a higher HR compared with the injured/untreated, injured/treated, and uninjured/treated groups early in the experiments (Figure 22). There was no significant difference in HR between the uninjured/treated and injured/treated groups. The mean arterial blood pressure (MAP) decreased in the early stages of the experiments before subsequently increasing gradually, peaking at approximately the time that the treatment begun before gradually decreasing again in all but the placebo/untreated sheep (Figure 23). The injured/treated groups had a consistently lower MAP compared with the other groups and there was a significant difference in MAP (p = 0.0058) between the uninjured/treated and injured/treated groups. The mean pulmonary artery pressure (MPAP) increased gradually, with the injured/treated group having a consistently higher MPAP (Figure 24). There was no significant difference in MPAP between the uninjured/treated and injured/treated groups. There was an initial, subtle increase in the central venous pressure (CVP) that peaked at approximately 1 hour post-injury followed by a decrease that stabilised at approximately 1 hour after the treatment was begun. Further, CVP levels in the injured/treated and placebo/untreated sheep were consistently higher and lower, respectively, in the course of the experiments.\n\nMixed venous oxygen saturation (SvO2) had a lower baseline before eventually rising to a relatively stable and higher level for the treated sheep, and a slightly lower level for the untreated sheep (Figure 26). The injured/untreated sheep maintained a consistently lower SvO2 compared with the other groups. Except for the placebo/untreated group, there was a decrease in continuous cardiac output (CCO) from baseline to approximately 1 hour after the treatment was begun (Figure 27). There was a significant difference (p = 0.0009) in CCO between the uninjured/treated and injured/treated groups with CCO in the treated groups increasing sharply before plateauing, particularly in the uninjured/treated group. There was also a subsequent gradual decrease in CCO in the injured/treated group. Stroke volume (SV) began to increase 1 hour after the treatment was begun for all groups, except for the injured/untreated group in which levels remained relatively constant (Figure 28). The SV in the injured/treated group began to decrease after 6 hours of treatment, while SV in the uninjured/treated and placebo/untreated sheep increased steadily before decreasing or levelling out after 12 hours or more of treatment. There were no significant differences in SV between the injured/untreated and placebo/untreated. The stroke volume index (SVI) began to increase 1 hour after the treatment was begun for all groups, except for the injured/untreated group, for which SVI remained relatively constant (Figure 29). The SVI in the injured/treated group began to decrease after 6 hours of treatment while SVI in the uninjured/treated and placebo/untreated groups increased before subsequently decreasing or levelling out after 12 hours or more of treatment. There were no significant differences in SVI between groups. While the cardiac index (CI) of the uninjured/treated and placebo/untreated groups remained relatively close to baseline levels (Figure 30), the CI of the injured/treated and injured/untreated groups declined gradually over the course of the experiments.\n\nAfter an initial increase in systemic vascular resistance index (SVRI) approximately 1 hour after treatment (Figure 31), SVRI began to decrease in all experimental groups before plateauing 12 hours after treatment, followed by a gentle increasing trend until the end of the experiments. The SVRI in the injured/treated group was consistently below that of the other groups during treatment while that of the injured/untreated group was correspondingly higher. There was no significant difference in SVRI between the groups. The pulmonary vascular resistance index (PVRI) remained close to baseline levels for all of the groups after 1 hour of treatment while that of the injured groups progressively increased and that of the uninjured groups remained lower with a subtle decrease after 6 hours of treatment (Figure 32). The PVRI in the placebo/untreated sheep remained close to baseline levels and the lowest throughout the course of the experiment. After a small peak attained at the beginning of the treatment, the right ventricular stroke work index (RVSWI) in the uninjured sheep gradually increased while that of the injured sheep decreased (Figure 33). There was a significant difference (p = 0.0196) in the RVSWI gap between the uninjured/treated and injured/treated groups. RVSWI in the placebo/untreated group remained high, while that of the injured/treated group was consistently the lowest. The left ventricular stroke work index (LVSWI) gradually increased in the uninjured/treated and placebo/untreated groups and plateaued 12 and 18 hours after treatment was begun, respectively, while LVSWI in the injured/untreated and injured/treated groups of sheep decreased and plateaued at 12 hours after treatment was begun and trended upward after 18 hours of treatment (Figure 34). LVSWI in the placebo/untreated group remained consistently higher than in the other groups while that of the injured/treated group was consistently the lowest.\n\nFollowing a decrease in the coronary perfusion pressure (CPP) from baseline in the smoke-injured sheep, there was a subsequent increase in this parameter within 5 minutes prior to a sustained decrease up to 18 hours of treatment, followed by another increase for the subsequent 6 hours (Figure 35). There was a significant difference in CPP (p = 0.0018) between the uninjured/treated and injured/treated groups and CCP in the placebo/untreated sheep remained relatively stable after an initial, subtle increase.\n\nThere was an initial subtle decrease in arterial oxygen content (CaO2) from baseline in all groups before a sustained increase in the injured/untreated group, a steady level in the placebo/untreated sheep, and a sharp trough in the injured/treated and uninjured/treated groups (Figure 36). Following the trough, the CaO2 of the injured/treated group gradually returned to baseline levels while that of the uninjured/treated group continued along a downward trend. There was a significant difference (p < 0.0085) in CaO2 between the uninjured/treated and injured/treated groups.\n\nThere was a slight decrease in the oxygen delivery index (DO2I) in all groups 1 hour after treatment before a further marked decrease, except for the placebo/untreated sheep (Figure 37). There was a significant difference (p = 0.0013) in DO2I between the uninjured/treated and injured/treated groups. The injured/treated group had the lowest DO2I compared with the other groups while the placebo/untreated sheep maintained the highest DO2I profile. The oxygen extraction index (O2EI) decreased in all groups before plateauing after approximately 6 hours of treatment. Further, there was a significant difference (p = 0.0247) in O2EI between the injured/treated and uninjured/treated groups (Figure 38). The O2EI in the injured/treated and injured/untreated groups was consistently lower and higher, respectively, compared with those of the other groups.\n\nThere was a variation in the volume of intravenous fluids administered to sheep in the different experimental groups. The injured/treated sheep had the highest fluid requirements, while the placebo/untreated sheep required the least (Figure 39). There was a significant difference (p < 0.0001) in fluid requirements between uninjured/treated and injured/treated sheep. The injured/untreated and injured/treated groups produced the least and most urine on average, respectively (Figure 40). There was no significant difference in urine output between the uninjured/treated and injured/treated groups.\n\nThere was a significant difference (p < 0.0001) in the amount of alfaxalone required between the uninjured/treated and injured/treated groups (Figure 41). The uninjured/treated group required more alfaxalone on average and the injured/untreated group required the least amount on average. Ketamine requirements differed between groups, with the injured/untreated group requiring the highest amount on average and the injured/treated group requiring the least (Figure 42). There was no significant difference in the quantities of ketamine required between the uninjured/treated and injured/treated groups, but there were significant differences in midazolam requirements (Figure 43), occurred between the uninjured/treated and injured/treated groups (p = 0.0067).\n\nThere were no significant differences in heparin infusion doses between the uninjured/treated and injured/treated sheep. Heparin requirements for the placebo/untreated group were the lowest (Figure 44). Activated clotting time increased sharply from baseline during pre-treatment and peaked after 1 hour of treatment, before decreasing sharply and plateauing (Figure 45). There were no significant differences in activated clotting time between groups.\n\nThere were significant differences in the ECLS pump speed (Figure 46), blood flow (Figure 47), and pressure differential (Figure 48) between the uninjured/treated and injured/treated groups. Further pump speed, blood flow, and pressure differential were significantly different (p = 0.0022, p = 0.0095 and p = 0.0041, respectively) between the two groups that received ECLS. These parameters in the uninjured/treated group were consistently higher than those of the injured/treated group.\n\nData were initially available in abstract form and subsequently as a full publication on inflammatory cell infiltration into the lung tissue with a trend toward increased lung injury in sheep that inhaled smoke, revealing damage to the bronchiolar lining and infiltration of inflammatory cells5,22–24.\n\n\nDiscussion\n\nThe results of this study agree with and confirm earlier preliminary observations that ECLS causes a decrease in pulmonary compliance (Figure 2) over time20. It was expected that the injured sheep would have relatively lower SpO2 readings (Figure 3) compared with the other groups because of episodes of hypotension with hypoxemia, which can affect pulse oximeter function25. The relatively low etCO2 in the injured sheep suggested that the sheep may have hyperventilated (Figure 4), the causes of which were evaluated with respect to the reactive oxygen species or superoxide dismutase activity by a team from the source study26,27.\n\nThe relatively low blood pH in the injured/treated sheep as depicted in Figure 5, suggested that the sheep tended to have metabolic acidosis, as the same group of animals also had low etCO2. This also implies that there was no respiratory component that contributed to the observed acidosis. The low pCO2 in the uninjured/treated sheep could be a result of hyperventilation and the high pCO2 in the injured/untreated sheep suggested that CO2 clearance was curtailed by injury (Figure 6).\n\nThe treatment of the sheep contributed to lung injury by causing deterioration of pO2 (Figure 7). The low pO2 translated to a low partial arterial oxygen pressure/inspired oxygen proportion (PaO2/FiO2 ratio), which was much worse in the injured sheep. This finding showed that ECLS contributed to the deterioration of the PaO2/FiO2 ratio in the injured/treated group of sheep, a novel finding that was also unexpected in the primary study (this has since been replicated in a more recent study9). It could also be argued from the data that perhaps VV ECMO was performed in a suboptimal manner, considering that the sheep oxygenation appeared to be less effective on ECMO than on the ventilator alone, but this remains to be explored further in future.\n\nFurther, the relatively higher levels of [Hb] in the injured sheep suggested that these animals could have been dehydrated as a secondary consequence of excessive fluid loss due to inflammation and increased vascular permeability28, despite intravenous fluid replacement (Figure 8). However, blood total protein and albumin levels, which are better predictors of dehydration in sheep29, were not measured.\n\nThe inverse decrease in FO2Hb (Figure 9) relative to FCOHb (Figure 10) following smoke injury was expected and is a finding that is in agreement with other studies28,30,31. It has recently been demonstrated that FCOHb is not correlated to the extent of lung injury28. The gradual decrease in MetHb (Figure 11) was probably caused by the enzymatic activity of methaemoglobin reductase32 and the higher Hct observed in the injured sheep (Figure 12) could have been due to dehydration because Hct was measured by an automated method.\n\nAs presented in Figure 14, the [Ca2+] was lower than the published normal level of 2.4 mmol/L33. Stress associated with yarding of the sheep and phosphorus imbalance in feed are the most likely suggested causes of low [Ca2+]34. Fasting the sheep for 24 hours prior to the experimental procedures could also have contributed to the relatively low [Ca2+].\n\nThe increase in Cl- beyond the normal range of 105–110 mmol/L33 during the experiments (Figure 15) suggests that the sheep may have developed respiratory alkalosis. Hyperventilation or metabolic acidosis resulting from sustained salivary loss of sodium bicarbonate that was more severe in the injured/treated group may have played a role in hyperchloraemia, because Cl- is known to replace HCO3- when the latter is lost from the body35,36. Baseline [K+] in all the sheep (Figure 16) was below the published normal range of 4–5 mmol/L33 and this relative hypokalaemia may have been related to low K+ in the diet37–39. The normal anion gap (Figure 17) with decreased HCO3- (Figure 21) confirmed the presence of hyperchloraemic acidosis in all but the placebo/untreated sheep. The cause of the hyperchloraemia was likely the prolonged administration of 0.9% NaCl.\n\nAlthough normal [Glu] in ruminants is usually lower than that for other species, its relative progressive increase in the injured sheep (Figure 18) may have been related to stress and severe pain associated with injury or the development of enterotoxaemia40,41. The relative increase in [Lac] beyond the reported normal range of 1–2 mmol/L in the injured sheep (Figure 19) suggested dehydration, trauma, and sepsis42. In particular, sepsis is a concern related to the sub-optimal rumen function, leading to loss of its buffer effect and an increase in the number of anaerobic bacteria with prolonged hypomotility, such as that which occurs during long-duration anaesthesia. Therefore, the increases in both [Glu] and [Lac] are consistent with severe injury.\n\nDotted lines represent one-sided margins of error bars.\n\n\nBody temperature\n\nBody temperature in the untreated groups gradually increased from baseline levels and plateaued at approximately after 6 hours of treatment, and remained higher than that for the treated groups (Figure 49). There was no significant difference in body temperature between the treated groups.\n\nThe elevated Base (ecf) above +2 mmol/L for most of the first 12 hours in the placebo/untreated and injured/untreated sheep suggested that the sheep were metabolically alkalotic40, before returning to normal levels (Figure 20). The relatively low Base (ecf) (less than −2 mmol/L) was consistent with loss of HCO3- and the tendency of developing metabolic acidosis40 in the injured/treated sheep. The marked decrease in [HCO3-] in the injured sheep was consistent with metabolic acidosis and was more severe in the injured/treated group, as illustrated in Figure 21, thereby suggesting that ECLS was a contributing factor.\n\nThe resting HR of sheep is 50–80 beats/min33. In a study that instrumented conscious sheep, the baseline heart rate was registered as 106 ± 9 beats/min43. In the present report, all of the sheep had a relatively high HR, thereby suggesting that stress and pain were contributing factors (Figure 22). The gradual decrease in HR during the course of the experiments was consistent with the effects of anaesthesia33.\n\nIn sheep, a mean arterial pressure below 60 mmHg indicates inadequate tissue perfusion33. Although the MAP values in the injured sheep were lower than for the uninjured sheep (Figure 23), MAP values were still within the published normal value of 70 mmHg33; the magnitude of injury was again a predictor of how low the MAP was. Another predictor for the severity of the injury was the mean pulmonary artery pressure, which was highest for the injured/treated sheep as illustrated in Figure 24. The baseline values for MPAP were higher than the 17 ± 1 mmHg reported in another study that used sheep for experiments43. The baseline CVP in all of the sheep in the present report was > 10 mmHg (Figure 25), which was much higher than the 5.5 ± 1.2 mmHg reported elsewhere43 in instrumented conscious sheep and a novel finding in this study. Thus, in this study, the severity of injury and treatment contributed to the CVP elevations found among the sheep.\n\nLower and upper bar limits correspond to minimum and maximum urine output, respectively.\n\nDotted lines represent one-sided error bars.\n\nDotted lines represent one-sided error bars.\n\nDotted lines represent one-sided error bars.\n\nThere was a benefit of ECLS treatment for SvO2, as it remained high for both the injured/treated and uninjured treated groups (Figure 26). The consistently low SvO2 in the injured/untreated group was expected because of the slightly reduced cardiac output in this group; however, this level of SvO2 was still higher than that reported in other studies43. Smoke injury was associated with a sustained decrease in cardiac output in all the sheep that were exposed to smoke. As in CCO changes (Figure 27), the SV (Figure 28), SVI (Figure 29) and CI (Figure 30) all had similar profiles for different groups, with the injured sheep having lower values. The decrease in SVRI in all of the sheep at a later stage in the experiments suggested that there was systemic vasodilation (Figure 31). In contrast, the increase in PVRI in the injured sheep suggested that vasoconstriction was caused by exposure to smoke injury (Figure 32). The exposure to smoke injury worsened both RVSWI (Figure 33) and LVSWI (Figure 34) while there was an increase in both parameters in the uninjured sheep. Reduced RVSWI is associated with poor functioning of the right ventricle44,45 and LVSWI is a reliable parameter for left ventricular function46.\n\nThe reduction in coronary perfusion pressure in the injured/treated—and to a certain extent the uninjured/treated sheep—suggested that ECLS contributed to the decrease in CPP, in addition to smoke injury (Figure 35). The CPP is an indicator of myocardial perfusion and has been proposed as a drug target during resuscitation47. The observations in the present study support the suggestion that CPP could be used to predict the severity of injury in sheep.\n\nFurther, the apparent increase in CaO2 in the injured sheep (Figure 36) could have been due to the relative increase in [Hb] secondary to dehydration as illustrated in Figure 8. The low DO2I in the injured/treated and uninjured/treated groups suggested that ECLS also contributed to this, in addition to smoke, based on the relatively higher DO2I in the injured/untreated sheep (Figure 37). Interestingly, the O2EI (Figure 38) had a comparable profile to that of the PaO2/FiO2 ratio and could also be used to predict the contribution of ECLS to smoke-related injury.\n\nThe smoke-injured sheep required considerable amounts of intravenous fluids (Figure 39) to compensate for the losses from pulmonary exudation and inflammation28,30. The mean urine production in all groups (Figure 40) was marginally lower than the published normal of 1.2 mL/kg/h33 but still considered to be within the acceptable range for this cohort of sheep. Moreover, the dosage of anaesthetic drugs used was considered adequate for the experiments (Figure 41, Figure 42 and Figure 43). In addition, heparin infusion (Figure 44) was indicated to prolong the activated clotting time (Figure 45) in order to minimise the risk of thrombosis during intravascular procedures48.\n\nThe reduction in the ECLS pump speed (Figure 46), flow (Figure 47), and pressure differential (Figure 48) could have resulted from systemic hypotension contributing to low amounts of blood to the pump. The ECLS was configured such that the centrifugal pump pulled blood from the inferior vena cava and returned it into the right atrium; therefore, if the circulating volume was low, the flow would decrease for a given pump speed and in this case, both rpm and flow would reduce. Centrifugal ECLS pumps are known to be preload dependent and afterload sensitive49, thereby making rpm and flows directly proportional to each other. The reason for the systemic hypotension remains undetermined. It is possible that an unknown pulmonary component or product produced in the smoke-damaged lungs of the sheep played a role. It must be noted that the body temperature of the sheep was generally within the physiological range (Figure 49).\n\nCertain observations regarding this study could affect the interpretation of red blood cell indices and their derivatives. For example, animals differ from humans in that estimated changes in plasma volume is preferably determined by changes in packed cell volume (PCV) or haemoglobin concentration and total plasma protein (TPP)50–52. Moreover, in animals, there is a wider range of normal PCV than TPP53. In critical care for domestic animals, the change in both PCV and TPP is most useful as a crude index of change in plasma volume54. A centrifuge that spins minute amounts of blood for rapid, cost-effective determination of PCV and TPP permits instant adjustments in an animal’s fluid needs. However, measurements of PCV and TPP were not conducted in the primary study. As with all data that are collected with different objectives, it was tedious to align certain time points with real-time observations made in the laboratory, particularly for data that was manually input. There was also no information regarding pre-anaesthetic blood tests.\n\nAn additional limitation of this study is related to the overall objective of providing useful information relevant to the sheep ECMO model in particular, and to the scientific community interested in large animal experimentation and veterinary medicine in general. Because the method of data collection method has not been validated across several laboratories or research groups, it is considered relatively preliminary and further validation studies are required. Moreover, the number of sheep (Figure 1) was low and this was particularly so in the injured/untreated and placebo/untreated groups, thereby preventing comparisons between the treated and untreated sheep. A further limitation is that cytokine levels, as predictors of lung injury, were not quantified. Using ELISA assays to quantify cytokine levels proved difficult and the cost was prohibitive in the present study, although subsequent efforts were made by a few members of the original research group in this regard5. It is partially for this reason that pioneering studies16 have been proposed for the development of proteogenomic assays as an alternative to ELISA—to learn from circulating markers of acute inflammation in injured sheep used as models of intensive care, to understand critical illness.\n\nOn another note, it can be argued that sheep ECMO data is already out there; however, cardiac function and haemodynamic data have not been reported in sufficient detail in the manner of this study. Also, this paper has many figures which some readers may see as being unfocused. The upside is that in recent times, academic journals now have sufficient capacity for lengthy, more informative and detailed reports, preferably with all data in one publication, rather than ‘salami slicing’ publications. In the literature related to this burn and smoke inhalation model lung, function has probably been reasonably well reported, but cardiac function is probably less so – which makes this report to stand out strongly. Lastly, it can be construed that observational control groups are too small (n=1 or 2). Putting out data from small groups and placing it in graphs with experimental groups with n=8 may be seen as misleading, but cutting these observations from the other two groups completely would be a disservice to science, considering that few animals as possible are used for research purposes nowadays to prove a point.\n\nNevertheless, although ECMO treatment has been demonstrated to contribute to, and exacerbate the deterioration of pulmonary pathology by reducing lung compliance and PaO2/FiO2 ratio in sheep studies5,9,23, the understanding of ECMO in respiratory life-support in human medicine continues to grow in a positive direction overall; moreover, there is evidence that it is useful as a life-saving treatment. These novel observations from sheep could help in understanding similar pathology such as that which occurs in animal victims of smoke inhalation from house or bush fires, aspiration pneumonia secondary to tick paralysis, and in the management of COVID-19 in humans55–57.\n\nThe World Health Organization (WHO) has recently recognised and classified COVID-19, caused by the novel coronavirus SARS-CoV-2, as a global pandemic and public health emergency58,59. The National Institute of Allergy and Infectious Disease (NIAID) of the United States of America recognises that coronaviruses constitute a large group of viruses known to cause respiratory diseases, including the common cold60. However, in recent years, three novel members of this family of viruses have arisen from animals to cause severe and extensive infection and death in humans61. In addition to bats, a large number of coronaviruses are known to circulate in certain domestic animals like cats and occasionally spill over to humans and cause serious illnesses, such as the SARS coronavirus (SARS-CoV) that emerged in Southern China in 200262. As a future perspective, the outcomes of the present study could be used to guide additional studies to enable the mortality indicators and prognostic indicators associated with ECMO and allied technology to be further evaluated and well understood in sheep and other experimental animals.\n\n\nConclusions\n\nThe results of this study demonstrated that ECLS contributed to the worsening of pulmonary pathology by reducing lung compliance and PaO2/FiO2 ratio. The O2EI changes mirrored those of the PaO2/FiO2 ratio and decreasing CPP was a predictor of a greater magnitude of cardiopulmonary injury in sheep. These novel observations could help in further understanding similar pathology in other patients; for example, in the resuscitation of animals injured from in house or bush fires. A similar data acquisition approach could be used in evaluating the effectiveness of a given experimental or clinical intervention to further the understanding of the clinical condition being studied and to aid in the formulation of treatments aimed at improving the survival of animal patients. In veterinary medicine, albeit now a considerably expensive and remote option, ECLS knowledge could complement the treatment of potentially reversible aspiration pneumonia, a secondary complication associated with both Ixodes holocyclus toxicity and laryngeal paralysis, in valuable companion animals and in critically ill humans who require respiratory support, like COVID-19 patients.\n\n\nData availability\n\nDryad: Cardioespiratory physiological perturbations after acute smoke-induced lung injury and during extracorporeal membrane oxygenation support in sheep. http://www.doi.org/10.5061/dryad.3r2280gd518.\n\nThis project contains the following underlying data:\n\nC24H-01.xls (24 hours of monitoring only no smoke inhalation and no ECMO).\n\nE24H-01 Saul.xls (24 hours of ECMO only).\n\nE24H-02 Saul.xls (24 hours of ECMO only).\n\nE24H-03 Saul.xls (24 hours of ECMO only).\n\nE24H-04 Saul.xls (24 hours of ECMO only).\n\nE24H-05 Saul.xls (24 hours of ECMO only).\n\nE24H-06 - 4146 Saul.xls (24 hours of ECMO only).\n\nE24H-07 Saul.xls (24 hours of ECMO only).\n\nE24H-08∀4630 Saul.xls (24 hours of ECMO only).\n\nE24H-Activated Clotting Time (Saul).xls (24 hours of ECMO only).\n\nE24H-Activated Clotting Time (Saul).xls (24 hours of ECMO only).\n\nE24H-Anaesthetics, Inotropes & Anticoagulants (Saul).xls (24 hours of ECMO only).\n\nE24H-Arterial Blood Gas Values (Saul).xls (24 hours of ECMO only).\n\nE24H-BP, ventilation & haemodynamic data (Saul).xls (24 hours of ECMO only).\n\nE24H-Calculated Resp+Haemodynamic Variables (Saul).xls (24 hours of ECMO only).\n\nE24H-Fluids and Urine Production (Saul).xls (24 hours of ECMO only).\n\nSC24H 1+2 PF and Carboxy.xlsx (24 hours of monitoring only after smoke inhalation, no ECMO).\n\nSC24H-01 Saul.xls (24 hours of monitoring only after smoke inhalation, no ECMO).\n\nSC24H-02 Saul.xls (24 hours of monitoring only after smoke inhalation, no ECMO).\n\nSE24H-01 Saul.xls (24 hours of ECMO after smoke inhalation).\n\nSE24H-02 Saul.xls (24 hours of ECMO after smoke inhalation).\n\nSE24H-03 Saul.xls (24 hours of ECMO after smoke inhalation).\n\nSE24H-04 Saul.xls (24 hours of ECMO after smoke inhalation).\n\nSE24H-05 Saul.xls (24 hours of ECMO after smoke inhalation).\n\nSE24H-06 Saul.xls (24 hours of ECMO after smoke inhalation).\n\nSE24H-07 Saul.xls (24 hours of ECMO after smoke inhalation).\n\nSE24H-08 Saul.xls (24 hours of ECMO after smoke inhalation).\n\nSE24H-Activated Clotting Time (Saul).xls (24 hours of ECMO after smoke inhalation).\n\nSE24H-ALL SO FAR (6) Saul.xls (24 hours of ECMO after smoke inhalation).\n\nSE24H-Anaesthetics, Inotropes & Anticoagulants (Saul).xls (24 hours of ECMO after smoke inhalation).\n\nSE24H-Arterial Blood Gases (Saul).xls (24 hours of ECMO after smoke inhalation).\n\nSE24H-Calculated Resp + Haemodynamic Variables (Saul).xls (24 hours of ECMO after smoke inhalation).\n\nSE24H-Fluids and Urine Production (Saul).xls (24 hours of ECMO after smoke inhalation).\n\nSE24H-Physiologic monitoring data (Saul).xls (24 hours of ECMO after smoke inhalation).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0). (https://creativecommons.org/licenses/by/4.0/legalcode).", "appendix": "Author contributions\n\n\n\nThe author (SC) was solely responsible for the study design, writing the manuscript, analysing and interpreting the data, and final approval of the manuscript. In addition, SC is fully accountable for the work.\n\n\nAcknowledgements\n\nGratitude is extended to members of the sheep ECMO research team at the Critical Care Research Group laboratory that is affiliated with The University of Queensland for their various roles during this research and the staff at QUT-MERF for assistance in the care of the sheep. The author is also thankful to Dr. Jane Charbonneau and SCRIBENDI for editorial support.\n\n\nReferences\n\nChemonges S, Shekar K, Tung JP, et al.: Optimal management of the critically ill : anaesthesia, monitoring, data capture, and point-of-care technological practices in ovine models of critical care. Biomed Res Int. 2014; 2014: 468309. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPlatts DG, Hilton A, Diab S, et al.: A novel echocardiographic imaging technique, intracatheter echocardiography, to guide veno-venous extracorporeal membrane oxygenation cannulae placement in a validated ovine model. Intensive Care Med Exp. 2014; 2(1): 2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPlatts DG, Diab S, Dunster KR, et al.: Feasibility of Perflutren Microsphere Contrast Transthoracic Echocardiography in the Visualization of Ventricular Endocardium during Venovenous Extracorporeal Membrane Oxygenation in a Validated Ovine Model. Echocardiography. 2015; 32(3): 548–556. PubMed Abstract \| Publisher Full Text\n\nShekar K, Fung YL, Diab S, et al.: Development of simulated and ovine models of extracorporeal life support to improve understanding of circuit-host interactions. Crit Care Resusc. 2012; 14(2): 105–111. 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(Australian and New Zealand Council for the Care of Animals in Research & Teaching, The University of Adelaide). 2009. Reference Source\n\nMcGrath SR, Lievaart JJ, Friend MA: Extent of utilisation of dual–purpose wheat for grazing by late–pregnant and lambing ewes and producer–reported incidence of health issues in southern New South Wales. Aust Vet J. 2013; 91(10): 432–436. PubMed Abstract \| Publisher Full Text\n\nHandy JM, Soni N: Physiological effects of hyperchloraemia and acidosis. Br J Anaesth. 2008; 101(2): 141–150. PubMed Abstract \| Publisher Full Text\n\nPeacock RE, Smart L: False hyperchloraemia in a dog secondary to ingestion of horse feed supplemented with potassium bromide. Aust Vet J. 2013; 91(8): 320–322. PubMed Abstract \| Publisher Full Text\n\nDuhm J, Göbel BO: Na+-K+ transport and volume of rat erythrocytes under dietary K+ deficiency. Am J Physiol. 1984; 246(1 Pt 1): C20–29. PubMed Abstract \| Publisher Full Text\n\nMien TXN, Yang LE, Fletcher NK, et al.: Effects of K+-deficient diets with and without NaCl supplementation on Na+, K+, and H2O transporters' abundance along the nephron. Am J Physiol Renal Physiol. 2012; 303(1): 92–104. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOh YT, Kim J, Youn JH: Role of pituitary in K+ homeostasis: impaired renal responses to altered K+ intake in hypophysectomized rats. Am J Physiol Regul Integr Comp Physiol. 2013; 304(12): R1166–1174. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRussell KE, Roussel AJ: Evaluation of the Ruminant Serum Chemistry Profile. Vet Clin North Am Food Anim Pract. 2007; 23(3): 403–426. PubMed Abstract \| Publisher Full Text\n\nSejrsen K, Hvelplund T, Nielsen MO: Ruminant physiology: digestion, metabolism and impact of nutrition on gene expression, immunology and stress. (Wageningen Academic Publishers, Wageningen). 2006. 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PubMed Abstract\n\nStafford KJ, Mellor DJ, Todd SE, et al.: The physical state and plasma biochemical profile of young calves on arrival at a slaughter plant. N Z Vet J. 2001; 49(4): 142–9. PubMed Abstract \| Publisher Full Text\n\nJacobs JP, Stammers AH, Louis JS, et al.: Extracorporeal Membrane Oxygenation in the Treatment of Severe Pulmonary and Cardiac Compromise in Coronavirus Disease 2019: Experience with 32 patients. ASAIO J. 2020; 66(7): 722–730. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamanathan K, Antognini D, Combes A, et al.: Planning and provision of ECMO services for severe ARDS during the COVID-19 pandemic and other outbreaks of emerging infectious diseases. Lancet Respir Med. 2020; 8(5): 518–526. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRajagopal K, Keller SP, Akkanti B, et al.: Advanced Pulmonary and Cardiac Support of COVID-19 Patients: Emerging Recommendations From ASAIO-A \"Living Working Document. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "70256", "date": "03 Sep 2020", "name": "Gabrielle C Musk", "expertise": [ "Reviewer Expertise veterinary anaesthesia", "respiratory physiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 provides extensive physiological data collected opportunistically during sheep studies of acute lung injury. The manuscript is well written and comprehensive. I have a few minor comments to address:\nInclude details on the fate of the animals at the end of the experiment.\n\nInclude brief details of the anaesthesia protocol in the materials and methods instead of just the reference to previous work.\n\nFinal paragraph page 13: suggest using the term nociception instead of pain as the animals were unconscious. Also tachycardia can occur with ketamine administration so may have contributed.\n\nFinal paragraph page 20: rephrase 'already out there' to 'previous published' or similar.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "69959", "date": "09 Sep 2020", "name": "Noah H Hillman", "expertise": [ "Reviewer Expertise Neonatal lung disease and sheep ventilator 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 manuscript provides information on the physiology from ECMO experiments in adult sheep. The article discussed everything from the physiology of the lung disease and the parameters used for the ECMO system. It gives some guidance for other researchers working on sheep models of ECMO or lung injury responses.\nThe information on the sedation drugs used will give guidance to other utilizing these therapeutic surgical options. Although mentioning what a research team used in an experiment is not entirely the same as proving these are the optimal treatment for the animals.\nThe conclusions in abstract are very broad, as it is unclear how a study on smoke inhalation helps with management of tick paralysis unless these animals get severe lung disease.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-769
https://f1000research.com/articles/9-768/v1	24 Jul 20	{ "type": "Research Article", "title": "Self-reported competency, knowledge and practices of teachers teaching primary children with autism in government schools of West Malaysia: a cross-sectional study", "authors": [ "Jo Ann Andoy-Galvan", "Sapna Shridhar Patil", "Prabhagari Nair Ramalingam", "Muhammad Aminul Syahmi Bin Shobri", "Karuthan Chinna", "Muhammad Sabri Sahrir", "Kumarappan Chidambaram", "Sapna Shridhar Patil", "Prabhagari Nair Ramalingam", "Muhammad Aminul Syahmi Bin Shobri", "Karuthan Chinna", "Muhammad Sabri Sahrir", "Kumarappan Chidambaram" ], "abstract": "Background: Disability brings challenges and demands in the family and society which last for a long time. Children that are affected by disability are often kept at home, without access to opportunities other children might have, and education is one of the most effective ways to break the cycle of discrimination and poverty. Malaysia is committed to achieving the Sustainable Development Goals, and teacher quality issues are among the Ministry of Education's focuses to ensure a successful journey for pupils with disabilities. In this study, we evaluated the competency, knowledge and implementation practices of teachers serving government schools in West Malaysia regarding teaching children with autism spectrum disorder (ASD). Methods: 832 primary teachers from different states of West Malaysia participated in a web-based survey that assessed self-reported competency, knowledge and implementation practices with regards to managing children with autism spectrum disorder. Results: Respondents rated themselves as quite competent, and believed that they were knowledgeable regarding ASD and could implement ASD tasks. Frequency of training was a consistent predictor of competency. Respondents who had never attended a training course had significantly lower self-competency, lower level of knowledge, and lower implementation ratings than those who had attended even one course. Conclusions: Investments in training teachers on ASD are highly recommended to ensure these students are provided with quality of education they deserve.", "keywords": [ "competency", "knowledge", "implementation", "autism", "teachers", "Malaysia", "government schools" ], "content": "Introduction\n\nDisability brings challenges and demands in the family and society which last for a long time. The financial burden associated with accessing health, education and social services is responsible for the cycle of poverty in society. Education is one of the most effective ways to break the cycle of discrimination and poverty that children with disabilities and their families often face by allowing them a greater degree of independence and better quality of life1. Disabled citizens are often underestimated for their sufficient capabilities to contribute to society resulting in not being prioritized. Education for All (EFA), a global movement, which aims to ensure that every child and adult receives basic education of good quality, has realized failure in its objectives because disabled children are not given proper attention. These children are often kept at home, without access to opportunities other children might have2. Universal primary education is one of the eight international Millennium Developmental Goals (MDGs). MDGs had been established following the Millennium Summit of the United Nations in 2000, following the adoption of the United Nations Millennium Declaration. It is a commitment made by 191 United Nation States to be achieved by the year 20153. According to the MDGs report in 2015, there has been a significant improvement in terms of universal primary education. The primary school net enrolment rate in the developing regions reached 91% in 2015, up from 83% in 2000. The number of out-of-school children of primary school age worldwide has fallen by almost half, to an estimated 57 million in 2015, down from 100 million in 2000. The literacy rate among youth aged 15 to 24 has increased globally from 83% to 91 % between 1990 and 20154. But despite impressive strides, there are more people being left behind, especially the poorest and those disadvantaged because of their sex, age, disability, ethnicity or geographic location2. Targeted efforts are required to reach the most vulnerable people. ‘Children with disabilities’ is one of these groups identified in the report as holding back progress towards education for all. Children with disabilities demand increased attention and often require an education adapted to their needs. Ensuring that all people have an equal chance of education must be at the heart of new goals post 2015.\n\nThe Sustainable Developmental Goals (SDGs) were developed to accomplish the unmet MDGs which ended in 20155. One SDG is to “ensure inclusive and equitable quality education and promote lifelong learning opportunities for all”6. This inclusive education is aimed at providing special education to students for them to be more successful in social interactions leading to further success in life. However, children with autism require specialized instructions and behaviour support to learn critical skills and improve long term outcomes7. The competency of teachers is important in the development of these special children. They should be well competent to ensure the quality of education that these children deserve.\n\nConsequently, this research evaluates the characteristics of teachers teaching children with autism in primary government schools in West Malaysia and their self-reported competency, level of knowledge and teaching practices implemented. The implications of this study are important as the results can impact the qualifications of teachers and guide professional development that will ensure potential development of children with ASD.\n\n\nMethods\n\nThis cross-sectional study, conducted in West Malaysia between June 2017 and January 2018, utilized a web-based survey to determine the level of competency, knowledge and implementation practices of primary government schoolteachers registered under the Program Pendidikan Khas Integrasi (PPKI) and Sekolah Kebangsaan Pendidikan Khas (SKPK). PPKI is a special education program for primary school children with disabilities, which was introduced by the Ministry of Education in 19628. It provides a separate class for disabled individuals organized under mainstream primary schools within integrated setting so they can interact and play with other children of their age. SKPK is a special education school provided by the same ministry, which provides education for primary school children with special needs. At present, there are 22 SKPK all over the country, which offer special education for different groups of disabilities; some exclusively provide education to deaf and mute children, while 12 offer programs for autism enrollees. Due to the scarcity of the available enrollees from SKPK (Table 1), regular government schools offering PPKI programs were also targeted (Table 2) in the present study.\n\nSource: Ketua Unit, Unit Data dan Maklumat, Cawangan Perancangan dan Penilaian, Bahagian, Pendidikan Khas, Kementerian Pendidikan Malaysia.\n\nSource: Ketua Unit, Unit Data dan Maklumat, Cawangan Perancangan dan Penilaian, Bahagian, Pendidikan Khas, Kementerian Pendidikan Malaysia\n\nThis survey was completed by teachers specifically teaching children with ASD employed in schools involved in SKPK and PPKI.\n\nInclusion criteria was primary schoolteachers presently working with children with autism during the study period.\n\nThis research adapted the same questionnaire used by Hendricks in the study ‘Skill competencies for professionals and paraprofessionals in Virginia supporting individuals with autism across the lifespan’9. This questionnaire was used to survey special education teachers employed in public schools in Virginia, USA The Virginia Skill Competencies is a list of guidelines for educators who serve students with autism and it was generated to develop educational standards in autism10.\n\nThe questionnaire consisted of four sections: Section A, background information, which comprised questions covering the following: age, gender, race, position, state, highest level of education, number of years spent/experience in teaching children with autism, number of ASD students taught in the last five years, number of students in a class, and frequency of special trainings attended; Section B, self-reported competency, which included nine statements on the self-rating of competence in handling a child with autism using a four-point Likert scale; Section C, self-rating of level of knowledge; and Section D, self-rating of level of implementation. These last two sections included statements organized under the following focus areas:\n\n1. General autism competencies\n\n2. Environmental structure and visual support competencies\n\n3. Comprehensive instructional programming competency\n\n4. Communication competencies\n\n5. Social skill competencies\n\n6. Behavior competencies\n\n7. Sensory motor development\n\n8. Independence and aptitude competencies\n\nSection C consisted of 16 statements on self-rating of level of knowledge and Section D included 25 statements on self-rating of the implementation practices. They used a five-point Likert scale when rating current level of knowledge, where 1 represented ‘little knowledge’ and 5 indicated ‘very knowledgeable’. When rating current level of implementation, 1 represented ‘rarely implemented’ and 5 indicated ‘frequently implemented’.\n\nThe questionnaire was translated into the Malay language by a professor teaching language studies. A pilot test was carried out to ensure validity and reliability of the questionnaire. Special Education Teachers at a private therapy center were asked to participate in the pilot survey and comment if the instructions were clear, comprehensive and easy to understand. They were also asked if the confidentiality was appropriately maintained in the questionnaire. Suggestions were collected and integrated into the questionnaires.\n\nThe link to the online survey was sent to the email address of the State Education Department directors of the 13 states. The researcher followed up using telephone calls to confirm receipt of the email. Jabatan Pendidikan Negeri (State Education Department) officers were assigned to coordinate with the researcher. The online questionnaire was administered, and data collected, using Google Forms.\n\nData was exported and analyzed using SPSS version 19. Frequencies, means and standard deviations were used to summarize the data. Linear regression procedures were conducted to determine whether certain socio-demographic variables would predict self-competence in ASD management, knowledge of ASD competencies, and knowledge of implementation of ASD competencies. For all tests, the level of significance was set at 0.05.\n\nApproval for this study was obtained from the Institutional Review Board of Taylor's University Centre for Research management and the Ministry of Education, Malaysia [KPM.600-3/2/3 Jld.3411; KPM.600-3/3/3 Jld.3537]. Written informed consent was obtained from the participants for participation in the study.\n\n\nResults\n\nOut of the 7,575 teachers teaching in West Malaysia (Table 3) in both SKPK and PPKI schools, 1073 responded and completed the survey (Table 4). A total of 242 responses were excluded due to missing information.\n\nPPKI, Program Pendidikan Khas Integrasi; SKPK, Sekolah Kebangsaan Pendidikan Khas.\n\nASD, autism spectrum disorder.\n\nAs per Nunnally and Bernstein (1994), a measure is moderately reliable if its Cronbach’s alpha is 0.70 or higher12. Given this criterion, the three measures (Sections B–D) were all reliable. Mean composites were created for each of the measures; as such, four was the highest possible score. As shown in Table 5, the sample of respondents rated themselves as considerably competent (M = 3.06, SD = 0.38). They also believed that they were knowledgeable regarding ASD competencies (M = 3.28, SD = 0.60) and could implement ASD tasks (M = 3.33, SD = 0.63).\n\nLinear regression procedures were conducted to determine whether certain socio-demographic variables would predict self-competence in ASD management, knowledge of ASD competencies, and knowledge of implementation of ASD competencies. Prior to conducting the regressions, several variables were recoded. First, level of education was recoded into a three-category variable: diploma, Bachelor’s, and graduate degree. Second, class size was recoded into a three-category variable: 1 to 5, 6 to 10, and 11 or more. Lastly, frequency of training was recoded into a four-category variable: never, once, once a year, and more than once a year. Indicator (or dummy) variables were then created, where the first category (i.e., diploma, 1 to 5, and never) served as the reference group. In addition, because the variable, number of ASD students taught, was severely skewed, this variable was transformed using natural log function. Skewness dropped to acceptable limits (i.e., skewness index fell below three, per Kline, 2011)13; thus, this transformed variable was used in subsequent regression procedures.\n\nAssumptions of multivariate normality (assessed through a normal probability plot), linearity, and homoscedasticity (both assessed through a plot of the studentized deleted residuals by the standardized predicted values) were checked; all assumptions were met. The problem of multi-collinearity was also verified via the variables’ tolerance values; multi-collinearity was not a problem as all tolerance values were above .2014\n\nThe findings in Table 6 reveal that the number of ASD students taught positively predicted self-competence ratings (β = .11, p = .003); the more ASD students the respondents had taught, the higher their self-competence ratings. Frequency of training also positively predicted self-competence ratings; respondents who had never attended a training course (M = 2.88, SD = .41) had significantly lower self-competency ratings than those who had attended even one course (M = 3.06, SD = .36; p < .001), those who attended a course once per year (M = 3.11, SD = .41; p < .001), and those who took more than one course a year (M = 3.11, SD = .34; p < .001).\n\nASD, autism spectrum disorder. Overall model F(9, 822) = 5.71, p < .001, R2 = .059. * p < .05. p < .01. * p < .001.\n\nThe findings in Table 7 show that the number of ASD students taught positively predicted knowledge of ASD competencies (β = .18, p < .001); the more ASD students the respondents had taught, the higher their knowledge of ASD competencies scores. Level of education also significantly predicted knowledge of ASD competencies; respondents with a teaching diploma had significantly lower knowledge ratings (M = 3.25, SD = .57) than respondents with a graduate degree (M = 3.43, SD = .68; p = .022). Frequency of training also positively predicted knowledge ratings; respondents who had never attended a training course (M = 2.95, SD = .71) had significantly lower knowledge of competency ratings than those who had attended even one course (M = 3.29, SD = .58; p < .001), those who attended a course once per year (M = 3.37, SD = .58; p < .001), and those who took more than one course a year (M = 3.34, SD = .55; p < .001).\n\nASD, autism spectrum disorder. Overall model F (9, 822) = 7.73, p < .001, R2 = .078. * p < .05. p < .01. * p < .001.\n\nThe findings in Table 8 indicate that number of ASD students taught positively predicted knowledge of implementation (β = .15, p < .001); the more ASD students the respondents had taught, the higher their knowledge of implementation scores. Level of education also significantly predicted knowledge implementation; respondents with a teaching diploma had significantly lower ratings (M = 3.21, SD = .59) than respondents with a graduate degree (M = 3.52, SD = .62; p = .001). Frequency of training also positively predicted implementation ratings; respondents who had never attended a training course (M = 2.99, SD = .77) had significantly lower implementation ratings than those who had attended even one course (M = 3.29, SD = .59; p < .001), those who attended a course once per year (M = 3.45, SD = .59; p < .001), and those who took more than one course a year (M = 3.40, SD = .58; p < .001).\n\nASD, autism spectrum disorder. Overall model F (9, 822) = 7.98, p < .001, R2 = .080. p < .05. p < .01. **p < .001\n\n\nDiscussion\n\nIncreasing numbers of autistic students in primary schools demand competent and well-trained teachers who can confidently implement efficacious strategies to bring the best possible outcomes. It is crucial to understand the aptitudes of special education teachers as their understanding of autism and its spectrum, strategies to design individual teaching plans and assessment will help the students achieve their potential. This study highlights the self-reported competency, knowledge and implementation practices and its predictors among special education teachers in primary government schools in West Malaysia. Additionally, the findings can serve as a guide to realigning the content of teachers’ preparation programs.\n\nIn our study, the majority of the participants had a Bachelor’s degree (83.3%) and another 8.2% had a Master’s degree. The average number of years of experience of teaching was 8.25 years, with an average of around 13 students with ASD, and majority of the teachers were experienced in handling a class size of 1 to 5. A similar profile was reported in studies conducted by Toran et al. and Wei et al.15,16). The teachers demonstrated intermediate to moderate levels of knowledge of autism and implementation practices. A study conducted in the city of Amman by AL Jabery et al.11 reported a similar result. However, our finding contrasted with finding of low to intermediate autism knowledge among special education teachers from the Virginia state of the United States of America as reported by Hendricks. A study conducted by Mavropoulou and Padeliadu among Greek educators reported and another by Toran et al. among teachers in Malaysia highlighted the low level of knowledge of autism among the special education teachers9,17,18.\n\nA moderate level of self-reported competency in determining appropriate intervention goals for the autistic children was reported by respondents in our study. Hendricks , Toran et al. and Wei et al. noted similar findings in their studies9,14,16.\n\nOur study revealed that the frequency of training strongly predicted self-reported competency and knowledge, as well as the implementation practices. Toran et al. emphasized the need to improve special education teacher training in order to increase the level of knowledge in autism and effective use of evidence-based teaching strategies by offering hands-on activities. Hendricks also highlighted the need for increased content related to autism and evidence-based practices during pre-service training9,15 Scheuermann et al. recommended the use of specialized skills training and provision of technical assistance and support to teachers in the training programs in the United States. Similarly, Litton et al. recommended a one-year induction program in conjunction with the local school districts’ programs with the express purpose of retaining highly qualified teaching personnel19,20.\n\nIn this study, we also found that the level of education also significantly predicted knowledge of ASD competencies and implementation practices. Respondents with a graduate degree had significantly higher ratings as compared to those with a teaching diploma. This finding highlights the importance of recruiting teachers with a higher level of education in this setting.\n\nThe number of students with ASD taught by the respondents was found to be a significant predictor of self-competence and knowledge, as well as the implementation practices. This is of particular interest given the challenges educators face due to the multidimensional nature of the disability. As documented by Hendricks9, the wide range of cognitive abilities and verbal skills of learners affect the knowledge and competencies required. Dealing with students with varying capabilities warrant special practices that address communication and social needs and help the learners achieve academic success.\n\nHowever, in our study the number of years of teaching experience did not predict self-competence or knowledge, which concurs with the finding reported by Alharbi et al.21. In contrast, AL Jabery et al.11 reported statistically significant differences in the level of knowledge among teachers with a teaching experience of five years and more as compared to those with a teaching experience of less than three years\n\n\nConclusion\n\nFrom our results, we conclude that teachers rated themselves as considerably competent. They also believed that they were knowledgeable regarding ASD and could implement ASD tasks. Frequency of training is a consistent predictor of this competency; respondents who had never attended a training course had significantly lower self-competency, lower level of knowledge and lower implementation ratings than those who had attended even one course. Investments in training of these teachers is highly recommended to ensure these students are provided with the quality of education they deserve.\n\n\nData availability\n\nHarvard Dataverse: Self reported competency, knowledge and practices of teachers teaching primary children with autism in government schools of West Malaysia, https://doi.org/10.7910/DVN/TP5VJI22.\n\nThis project contains the following underlying data:\n\n\n\n- Raw data: ASD TEACHERS.tab\n\nHarvard Dataverse: Self reported competency, knowledge and practices of teachers teaching primary children with autism in government schools of West Malaysia, https://doi.org/10.7910/DVN/TP5VJI22.\n\nThis project contains the following extended data:\n\n- Questionnaire in English and Malay language (competency developed by Virginia Autism Council)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nThe authors would like to thank the following people from the Ministry of Education for their support to this research project: Shazali Bin Ahmad, Rosli Bin Ismail, Mohd Helmy Bin Mahat, Fatimahtul Zaharah binti Ithnin; and all the state Education Offices for their assistance in the data collection particularly the following officers: Mohamad Faizal bin Mohamad Roselee, Nawi Awang Senik, Hj Mohd Nazri Bin Jj Abdul Latip, Wan Shariza Binti Ahmad, Shafruddin Ali Hussin, Huzaimah binti Muain, Yahaya Ali, Letchumy A/P Sinnan and Fakhrul Nizam Pakhruddin. Also thanks go to the principals, assistant principals, special education head and teachers of the Sekolah Kebangsaan Pendidikan Khas (SKPK) and Sekolah Kebangsaan (SKs).\n\n\nReferences\n\nUNICEF: The right of children with disabilities to education: A rights-based approach to Inclusive Education. Geneva: UNICEF Regional Office for Central and Eastern Europe and the Commonwealth of Independent States (CEECIS); [cited 2020 Jan 15]. 2012; 119. Reference Source\n\nUNESCO Education for all Global Monitoring Report 2015: Education for all 2000-2015: Achievements and Challenges. France: UNESCO Place de Fontenoy; [cited 2020 Jan 17]. 2015; 516. Reference Source\n\nWorld Health Organization: Millennium Development Goals (MDGs). Who.int.; [cited 2020 Jan 14]. 2015. Reference Source\n\nUnited Nations: The Millennium Development Goals Report 2015. New York: United Nations; [cited 2018 Feb5 15]. 2015; 72. Reference Source\n\nUnited Nations: The Sustainable Development Agenda - United Nations Sustainable Development. United Nations Sustainable Development. [cited 2020 Jan 14]. Reference Source\n\nUnited Nations: SDG Indicators. Unstats.un.org.; 2019. Reference Source\n\nWong C, Odom SL, Hume KA, et al.: Evidence-Based Practices for Children, Youth, and Young Adults with Autism Spectrum Disorder: A Comprehensive Review. J Autism Dev Disord. 2015; 45(7): 1951–66. PubMed Abstract \| Publisher Full Text\n\nMinistry of Educaton: Program Pendidikan Khas Integrasi. moe.gov.my. [cited 9 January 2020]. 2019. Reference Source\n\nHendricks D: Special education teachers serving students with autism: A descriptive study of the characteristics and self-reported knowledge and practices employed. J Vocat Rehabil. 2011; 35(1): 37–50. Publisher Full Text\n\nVirginia Autism Council: Skill competencies for professionals and paraprofessionals in Virginia supporting individuals with autism across the lifespan. Skill competency committee of the Virginia autism council. 2010; 1–30. Reference Source\n\nAL Jabery MA, Melhem AM, Al Abdallat BM: Level of Knowledge about Autism Disorder among Special Education Teachers Who Teach Individuals with Autism in the City of Amman. Dirasat Educational Sciences. 2014; 41(2): 881–99. Reference Source\n\nNunnally JC, Bernstein IH: Psychometric theory (3rd ed.). New York, NY: McGraw-Hill, Inc. 1994. Reference Source\n\nKline RB: Principles and practice of structural equation modeling. 3rd ed. New York: The Guilford Press. 2011; 427. Reference Source\n\nNorušis MJ; SPSS Inc: SPSS for Windows Base System Users Guide. Release 5.0. Englewood Cliffs: Prentice Hall. 1992; 672. Reference Source\n\nToran H, Westover JM, Sazlina K, et al.: The Preparation, Knowledge and Self Reported competency of Special Education Teachers Regarding Students with Autism. J Soc Sci Hum. 2016; 24(1): 185–96. Reference Source\n\nWei L, Yasin MHM: Teacher Training to Increase Teacher’s Competency in Teaching Autism Child. Journal of ICSAR. 2017; 1(1): 1–5. Publisher Full Text\n\nMavropoulou S, Padeliadu S: Greek Teachers’ Perceptions of Autism and Implications for Educational Practice. Autism. 2000; 4(2): 173–83. Publisher Full Text\n\nToran H, Yasin MHM, Tahar MM, et al.: Level of Training, Knowledge and Confidence of Special Need Teachers on Autism. Malaysian Journal of Education. 2010; 35(1): 19–26.\n\nScheuermann B, Webber J, Boutot E, et al.: Problems with Personnel Preparation in Autism Spectrum Disorders. Focus Autism Other Dev Disabil. 2003; 18(3): 197–206. Publisher Full Text\n\nLitton FW, Rotatori AR, Coombs-Richardson R, et al.: Preparation for Teachers for Students with Autism Spectrum Disorders: A Call for Quality and Quantity. Am J Educ Res. 2017; 5(2): 225–30. Reference Source\n\nAlharbi KA, Alharbi AA, Al-Thunayyan FS, et al.: School's Teachers Knowledge About Autism in Al-Badayacity, Al-Qassim Region, Kingdom of Saudi Arabia. Mater Sociomed. 2019; 31(1): 4–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGalvan JA: \"Self Reported Competency, Knowledge And Practices Of Teachers Teaching Primary Children With Autism In Government Schools Of West Malaysia\". Harvard Dataverse. V1, UNF: 6:AcZAHui+pvQ/O3/yya81RQ== [fileUNF]. 2020. http://www.doi.org/10.7910/DVN/TP5VJI" }	[ { "id": "69158", "date": "01 Oct 2020", "name": "Ivan Soldatovic", "expertise": [ "Reviewer Expertise Clinical Studies", "Epidemiology", "Public Health", "Biostatistics", "Internal medicine" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is interesting and provides information about self perceived competency regarding the work with autistic children. However, there are some concerns regarding the article.\n\nAbstract The Background is longer than Results and Conclusion. I suggest to make more interesting facts (or numbers) in Results than to write long introduction.\n\nIntroduction The last sentence contains only abbreviation (ASD).\n\nMethods Please make clearer about SKPK and Table 1 and . In text the authors states that 22 SKPK (12 autism) is present and in the Table 1 total number of SKPK is 53. Does it mean that total number of schools enrolled in this research is 53 SKPK + 1973 PKI? This part is somehow confusing due to specificity of the country educational organization. Authors might make it more simpler. It is important to emphasize the number of schools and number of teachers in each type of school that are enrolled in the research. Table 1 and 2 might be more appropriate as supplement since distribution of schools in the country is not so important information to the international readers, but rather specific for Malaysia. The authors state that they use adapted questionnaire. What adaption did the authors made? How many participants were used for the pilot test for validity and reliability? The authors should provide at least some information about that validation process of the questionnaire, for example (Cronbach’s alpha of scales, or at least interval of alpha levels for scales B-D 0.888-0.900).\n\nResults The author should present the percentage of total response rate and rate of teachers enrolled in the study after excluding missing. It is more informative then just counts. In discussion the authors must explain this low percentage. In Table 4, do the authors need to present each state? Does it change their discussion? The table is big and it would be easier to collapse some categories (states) to “Other”. The Overall procedure is confusing. Why did the authors made all numerical variables to binary or ordered. Linear regression is assumed to have numerical and categorical variables (binary or ordered) as independent variables. Did the authors made any model with original variables and concluded that this models are better? There is no univariable analysis. How did the authors choose these independent variables for the modeling? Why did not use Gender or others variables presented in Table 1? The authors might try (not necessarily) to merge Tables 6, 7 and 8 into one table with the predictors in rows (as it is) and three outcomes in column. It might provide better insight into relationship between independent predictor as specific outcome. Table 5 has alpha levels. Is Cronbach’s alpha=0.98 somehow too large? Some authors suggests that.\n\nDiscussion The authors repeat numbers from results (no need for that). First the response rate should be explained. It seems that is low and do the authors think that the sample is representative. In some paragraphs the authors leave out “self perceived” or “self-reported”. It is necessarily to emphasize that their knowledge and competency is self perceived, not completely objective (if I understood the questionnaire completely).\n\nIs the work 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? Partly", "responses": [] }, { "id": "72000", "date": "12 Oct 2020", "name": "Jihan A.Mostafa", "expertise": [ "Reviewer Expertise Neurology", "Neuroscience", "Psychiatry." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 cross sectional study clearly address the research question via proper methodology which explores the measurement of knowledge, competency, implementation of school teachers regarding ASD. However, choosing this survey which is a qualitative one (self- rating) is considered less choice than having a quantitative objective measurement of knowledge, competency especially there is other questionnaires present ( Autism Knowledge Questionnaire with 30 questions).\n\nRegarding predictors of competency: number of students ASD is considered a statistically significant one however, it is not mentioned in the abstract or the conclusion p< 0.05.\n\nPlease mention p value in the abstract for the training as a predictor.\n\nProper designing of the study with good methodology.\n\nGood editing and construction of the statistical analysis which was done in a proper way.\n\nThe authors clearly tested all predictors for competency, and knowledge in school teachers in proper analysis, but at the end they did not provide a detailed explanation for enhancement strategy for educational programs, program design and ways of evaluating these programs, quantitative measurement for an effective educational practice.\n\nThe authors neglected the influence of student support teams in a multidisciplinary manner and family-school collaboration.\n\nSample size is adequate however sampling technique is not that good.\n\nFinally, I think this study is interesting and deserves indexing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-768
https://f1000research.com/articles/9-634/v1	22 Jun 20	{ "type": "Research Article", "title": "Effect of supraneural transforaminal epidural steroid injection combined with caudal epidural steroid injection with catheter in chronic radicular pain management: Double blinded randomized controlled trial.", "authors": [ "Sithapan Munjupong", "Wipoo Kumnerddee", "Wipoo Kumnerddee" ], "abstract": "Background: Epidural steroid injection (ESI) has been used in managing chronic radicular pain. Regarding various techniques of ESI, the synergistic effect of caudal ESI (CESI) on transforaminal ESI (TFESI) in chronic lumbosacral radicular pain in prospective randomized controlled trial has not been determined. Methods: A total of 54 eligible patients with lumbosacral radicular pain were randomly allocated to undergo TFESI plus CESI (TC group) or TFESI alone (T group). The effective response to treatment was predefined by at least a 50% reduced verbal numerical rating scale (VNRS) from baseline between group comparison and the functional outcomes as measured by improved Oswestry Disability Index by least 15 points from baseline. All participants were evaluated using a single blinded outcome assessor before the procedure and at 1, 3 and 6 months after the procedure. P <0.05 was considered as statistically significant. Results: Average VNRS reduced significantly from baseline after receiving procedure at 1, 3 and 6 months in both groups (P-value <0.05). However, the TC group showed significant pain relief compared with the T group in spondylolisthesis and failed back surgery syndrome at 1 month. No statistical difference was observed between group comparisons of functional outcomes. Conclusions: A treatment combining TFESI and CESI showed significant pain relief over TFESI alone in spondylolithesis and failed back surgery syndrome at 1 month. No effect was found concerning functional evaluation. Registration: Thai Clinical Trials Registry ID TCTR20171101002 01/11/2017", "keywords": [ "Transforaminal", "caudal", "epidural steroid injections", "lumbosacral radicular pain", "randomized controlled trial." ], "content": "Introduction\n\nChronic lumbosacral radicular pain (CLRP) is a common condition in pain and spine centers. Treatment is challenging among patients who do not respond to either medication or physiotherapy and epidural steroid injection (ESI) is one commonly used intervention to alleviate radicular symptoms1. These inhibit the synthesis of prostaglandins, interrupting nociceptive c fibers and reducing edema surrounding the nerve root2–4.\n\nDifferent approaches of ESI are available, namely, transforaminal ESI (TFESI), interlaminar ESI (ILESI), and caudal ESI (CESI)5–9. The effectiveness of these three injected approaches has been shown. Related studies10–12 have reported that TFESI was more beneficial than CESI regarding pain relief in herniated disc or radicular pain13. However, one recent systematic review and meta-analysis revealed TFESI could be weakly recommended over CESI14. Furthermore, one retrospective study showed adjunctive CESI on TFESI significant relieved more pain than only TFESI15. Unfortunately, a prospective study has not been conducted of the synergistic effects of combining the epidural steroid approach. Consequently, this study aimed to compare the effectiveness of additional CESI to TFESI and TFESI separately in chronic lumbosacral radicular pain in a prospective randomized study and also to investigate possible complications during injection.\n\n\nMethods\n\nThe study comprised a prospective, single center, randomized, double blind, active-controlled parallel group. Permission to conduct this study was granted by the Institutional Review Board of the Royal Thai Army Medical Ethics Committee and registered with the Thai Clinical Trials Registry on 11 November 2017 (TCTR20171101002).\n\nThis study was conducted from November 2017 to January 2019. In total, 54 patients who met inclusion criteria were recruited. Patients attending the PMK Pain Treatment Center, Phramongkutklao Hospital were informed by a nurse anesthetist about the study. Patients who indicated an interest then provided written informed consent. The inclusion criteria comprised patients aged 18 to 80 years old with a history of chronic lumbosacral radicular pain (longer than six months) having a diagnosis of either symptomatology or physical examination correlated using magnetic resonance imaging (MRI) and unsatisfactory pain control with either medication or physiotherapy. The exclusion criteria comprised patients presenting significant neurological deficit or cauda equina syndrome, no absolute contraindications to intervention from MRI such as discitis or spinal infection, coagulopathy, psychiatric problem, pregnancy, language barrier or history of allergy to local anesthetics, triamcinolone and contrast media.\n\nAll patients were interviewed and physically examined by only one pain physician. Pain characteristics were documented and randomly allocated in two groups equally using computer-generated table and concealed envelope. The random numbers were kept sealed and opened by a nurse anesthetist uninvolved in this study. Those in the TC group received CESI in addition to TFESI, and the T group received only TFESI. All participants and one nurse anesthetist, trained as outcome assessor were blinded to the study group.\n\nThe intervention was performed as a day surgery using local anesthesia. The treatment level determined for supraneural transforaminal approach was based on clinical symptoms correlating with MRI. All patients were placed in the prone position, then pulse oximetry and noninvasive blood pressure were monitored. The lower back and buttocks areas were cleaned using sterile fashion technique.\n\nA C-arm fluoroscope (9900 Elite, Super C, OEC, UT, USA) was adjusted and rotated obliquely 20 to 25o ipsilateral to the affected side and 0 to 10o cephalo-caudad tilt until aligned with the superior vertebral end plate. The needle entry points were identified and the skin was infiltrated with 4 to 6 mL 1% lidocaine. A Quinke needle (22-G, 10 cm long) (Unilever, Japan) was inserted in the direction of the radiation beam. The supraneural transforaminal technique was performed, and the tip of the needle was placed below the pedicle and within the upper half of the intervertebral foramen in the lateral image. Then 0.5 to 1 ml of nonionic contrast media (Omipaque 300, GE Healthcare, Shanghai, China) was injected via extension tubing to confirm the needle’s location at the target area under real time fluoroscopy. The caudal epidural space was identified using fluoroscopic guidance in the lateral position and then a 16-gauge introducer Touhy needle (Epimed International RK, Epimed International, Johnstown, NY, USA) was inserted through the sacral hiatus into the caudal space and an epidural catheter was inserted using Touhy needle. Then 0.5 to 1 ml of nonionic contrast media was injected via epidural catheter (Epimed International RK, Epimed International, Johnstown, NY, USA) under real time imaging to confirm the desirable vertebral level and covered target site on both groups. T-group underwent 0.08% Levobupivacaine (Abbvie S.r.l., Italy) plus 40 mg of triamcinolone (L.B.S. Laboratory Ltd., Bangkok, Thailand) in a total volume of 3 ml via only the intervertebral foramen. Those in the TC group underwent 3 ml of 0.08% Levobupivacaine plus 40 mg of triamcinolone using the transforaminal approach combined with 10 ml of 0.025% Levobupivacaine plus 40 mg of triamcinolone via caudal epidural catheter.\n\nThe primary outcome was an effective response to treatment, predefined by at least a 50% reduced verbal numerical rating scale (VNRS; 0-100) from baseline16 between group comparisons. The secondary result was functional outcome, measured by improved Oswestry Disability Index (ODI, Thai version 1)17 at least 15 points from baseline. All participants were completely supervised and evaluated by blinded outcome assessor before the procedure, and then subsequently at 1, 3 and 6 months after procedure when attending the outpatient department of PMK Pain Treatment Center.\n\nThe sample size was calculated based on the related study of Ploumis A et al.12. The probability of significantly reduced pain from TFESI was 0.90, whereas the probability of significantly reduced pain from CESI was 0.545. The result indicated 24 patients were required for each group to reach a significance level of 0.05, the power of study was set at 80%, and we added 10% more for loss to follow-up. The final number of participants totaled 27 per group. All analyses and summaries were performed with Stata, Version 13/SE (StataCorp, 2013, College Station, TX, USA). A P value <0.05 was considered statistically significant. Descriptive statistics for continuous variables was presented as mean and standard deviation (SD) for sufficiently normally distributed variables. For nominal data, absolute and relative frequencies were displayed for each category. Independent t-test and Chi-square test were performed to compare the differences between groups in continuous variables and categorical variables, respectively. Multilevel mixed linear regression was performed to compare the Verbal Numerical Rating Scale (VNRS) and ODI Questionnaire change over the study period between both groups. A random intercept for the patients was included in the model to account for the cluster structure of the data (two-level models).\n\n\nResults\n\nIn total, 54 eligible patients were enrolled and allocated in equal groups of 27. Two participants were lost to follow-up due to progressive weakness and scheduled for surgery in the T group. Consequently, 25 patients remained in the T group and 27 in the TC group as shown in Figure 1. No differences were found regarding demographic data, sex, weight, clinical diagnosis, level of pain dermatome, preprocedural VNRS and ODI before intervention as presented in Table 118. Mean baseline VNRS was high in both groups; 74.8 ± 16.8 and 69.6 ± 15.1 in the T and TC groups, respectively.\n\nOverall, average VNRS was significantly reduced from baseline after receiving the procedure at 1, 3 and 6 months in both groups (P-value <0.05). However, the study showed significant differences between group comparisons at 1 and 3 months (P-value=0.009 and 0.044), respectively) as shown in Figure 218. Moreover, average ODI was significantly improved from baseline at 1, 3 and 6 months in both groups. Nonetheless, no significant difference was found in average ODI over the study period between group comparisons (p=0.235) as presented in Figure 318.\n\nThe number of patients, responding to treatment, was measured by decrease in VNRS of 50% or greater from baseline at each follow-up period, as presented in Table 218. No difference was found between group comparisons. However, subgroup allocation divided by etiologies found the TC group exhibited more patient responses to the procedure and a significant difference between groups in spondylolisthesis and failed back surgery syndrome at 1 month follow-up (p=0.005 and p=0.022, respectively) (Table 318). Moreover, the TC group were more satisfied with the treatment outcome than the T group in VNRS at 3 months for spondylolisthesis and failed back surgery syndrome, although not statistical significance (p=0.109 and p=0.154, respectively). Likewise, the study found no significant difference between two groups in herniated disc and spinal stenosis.\n\nVNRS=Verbal numerical rating scale, TFESI = transforaminal epidural steroid injection, T Group = transforaminal, TC group = transforaminal and caudal\n\n*Statistically significant VNRS=Verbal numerical rating scale, T Group = transforaminal, TC group = transforaminal and caudal\n\nFunctional outcome assessed by the number of patients with improved ODI at least 15 points at each follow-up period showed no significant difference between groups, classified by either radicular symptoms or etiology as shown in Table 4 and Table 518.\n\nTFESI = transforaminal epidural steroid injection\n\nT Group = transforaminal, TC group = transforaminal and caudal\n\nAdditionally, no serious complications, such as neurological deficit, was reported during the course of the study.\n\n\nDiscussion\n\nThis constituted the first prospective study to compare clinical outcomes of combined CESI to TFESI (TC group) and TFESI alone (T group) to alleviate chronic lumbar radicular pain from different etiologies such as herniated disc, spinal stenosis, spondylolisthesis and failed back surgery syndrome for which each degenerative spine disease may present distinctly varied responses from different ESI techniques.\n\nThis study showed combined CESI with TFESI provided more effective pain relief than TFESI separately among patients with spondylolisthesis and failed back surgery in which prior studies reported the mechanism of radicular pain in spondylolisthesis was usually from mechanical compression resulting in inflammatory changes in the enclosing nerve root and venous and arterial flow disability19,20. Accessing the epidural space of the supraneural TFESI is relatively difficult in a severely degenerated and narrowed foramen13,21. Moreover, the injected volume of lumbar TFESI is likely to influence the results. Prior studies have reported that larger injected epidural volumes provide effective pain relief16,22 and a larger injected volume can lavage waste products from the epidural space, reducing the abnormal signal of the offending nerve, and increasing blood flow to the ischemic nerve23. Desai et al.24 confirmed that the more vertebrae covered by the injected volume the better the outcome, and Furman et al.25 commented that a larger injected volume was needed for failed back surgery patients. Unsurprisingly, the TC group showed significant pain relief in spondylolisthesis and failed back surgery syndrome.\n\nUnfortunately, this study showed the TC group experienced more significant pain relief than the T group only at 1 month among patients with spondylolisthesis and failed back surgery syndrome. However, a trend was shown for higher pain relief in the TC group at 3 months (5 of 7 patients in the TC group compared with 2 of 7 patients in the T group) and 6 months (2 of 6 patients in the TC group compared with none of the patients in the T group), without significant difference. As described above, this might have been from the effect of combined techniques peaking at 1 month, then it might have gradually worn off from instability in spondylolisthesis and the return of softened epidural adhesion and fibrosis15,22 for which the epidural scar reduced the effectiveness of injection26.\n\nRecently, a retrospective study reported combined caudal and TFESI in herniated disc provided more significant pain relief and improved patient satisfaction than only TFESI at 1 year15. In contrast, this study showed no significant pain relief between the 2 groups, for which demographic data of our patients with herniated disc showed lower average ages (mean age 37 ± 8.5 and 35 ± 5.4 in the T and TC groups, respectively) than a related study (mean age 62.4 ± 15.5 and 57.6 ± 15.7 in the T and TC group, respectively) in which younger patients might have received greater benefit from steroid injection in accordance with Park et al.26 showing younger age produced a better response from TFESI. However, no significant difference was observed. Moreover, this study investigated just mild degree herniated disc such as mild unilateral paracentral disc herniation or mild foraminal stenosis and mild degree of spinal stenosis in which only the TFESI technique was sufficient to alleviate pain. Furthurmore, this study injected a larger volume in the T group (3.0 ml) compared with 1.5 ml in the study of TFESI by Kircelli A et al.15 in which a larger injected volume could cover more pain generators across multiple levels of the spine in accordance with Furman et al.25,27,28\n\nIn addition, this study postulated that synergistic anti-inflammatory effect from the double dose of steroid in the TC group may have conferred better pain relief. However, one related study reported 40 mg was as effective as 80 mg of methylprednisolone in TFESI for lumbar radicular pain29, while Kang et al.30 revealed no significant difference between 10, 20 and 40 mg of triamcinolone at 1 week in TFESI for disc herniation with lumbosacral radicular pain. Unsurprisingly, this study also showed no significant difference in herniated disc concerning different dosages of corticosteroid between the 2 groups.\n\nOur study had some limitations. Firstly, we demonstrated radicular pain from symptomatology and physical examination, for which the source of pain may have overlapped the pain referred pattern from the zygophysial joint, sacroiliac joint pain or enclosing soft tissues31–33, which might have limited the efficacy of the procedure. However, many common problems are involved in chronic low back pain. Secondly, electrodiagnosis was not performed in this study. Nonetheless, electrodiagnosis may have demonstrated false-negative findings, as demonstrated in a similar publication which showed 40 to 85% sensitivity depending on the referral range34,35. Thirdly, the study did not verify anterior or posterior epidural space of contrast flow which might have affected the efficacy of the result. Fourthly, this study included a small sample size for subgroup allocation which might not have been able to detect differences between groups. A larger sample size in each etiology should be demonstrated in further study. Lastly, we did not collect the data of oral medication. Further research is needed to determine whether combination of oral medication, physiotherapy and psychological effect.\n\n\nConclusion\n\nThe synergistic effect of CESI to TFESI was more effective than TFESI separately in spondylolisthesis and failed back surgery syndrome with no neurological deficit.\n\n\nData availability\n\nFigshare: data_epidural_04042020.xls. https://doi.org/10.6084/m9.figshare.12320846.v218\n\nThis project contains the following underlying data:\n\n- data_epidural_04042020.xls (Pain and disability index data for study participants. Data dictionary provided as extended data36)\n\nFigshare: Information of abbreviation data set. https://doi.org/10.6084/m9.figshare.12361499.v136\n\nThis project contains the following extended data:\n\n- Information of abbreviation data set.docx (Data dictionary)\n\nFigshare: CONSORT checklist for ‘Effect of supraneural transforaminal epidural steroid injection combined with caudal epidural steroid injection with catheter in chronic radicular pain management: double blinded randomized controlled trial’ https://doi.org/10.6084/m9.figshare.1236149037", "appendix": "Acknowledgments\n\nWe acknowledge Dr. Nattaya Udomsukdi for visualizing and collecting data and Mrs. Pimrapat Tengtrakulcharoen for her statistical advice and data analysis.\n\n\nReferences\n\nManchikanti L, Boswell MV, Singh V, et al.: Comprehensive evidence-based guidelines for interventional techniques in the management of chronic spinal pain. Pain Physician. 2009; 12(4): 699–802. PubMed Abstract\n\nKantrowitz F, Robinson DR, McGuire MB, et al.: Corticosteroids inhibit prostaglandin production by rheumatoid synovia. Nature. 1975; 258(5537): 737–9. PubMed Abstract \| Publisher Full Text\n\nFukusaki M, Kobayashi I, Hara T, et al.: Symptoms of spinal stenosis do not improve after epidural steroid injection. Clin J Pain. 1998; 14(2): 148–51. PubMed Abstract \| Publisher Full Text\n\nJohansson A, Hao J, Sjolund B, et al.: Local corticosteroid application blocks transmission in normal nociceptive C-fibres. Acta Anaesthesiol Scand. 1990; 34(5): 335–8. PubMed Abstract \| Publisher Full Text\n\nManchikanti L, Boswell MV, Datta S, et al.: Comprehensive review of therapeutic interventions in managing chronic spinal pain. Pain Physician. 2009; 12(4): E123–98. PubMed Abstract\n\nAtcheson SG, Dymeck T: Rapid resolution of chronic sciatica with intravenous infliximab after failed epidural steroid injections. Spine (Phila Pa 1976). 2004; 29(12): E248–50. PubMed Abstract \| Publisher Full Text\n\nManchikanti L, Abdi S, Atluri S, et al.: An update of comprehensive evidence-based guidelines for interventional techniques in chronic spinal pain. Part II: guidance and recommendations. Pain Physician. 2013; 16(2 Suppl): S49–283. PubMed Abstract\n\nPinto RZ, Maher CG, Ferreira ML, et al.: Epidural corticosteroid injections in the management of sciatica: a systematic review and meta-analysis. Ann Intern Med. 2012; 157(12): 865–77. PubMed Abstract \| Publisher Full Text\n\nManchikanti L, Benyamin RM, Falco FJ, et al.: Do epidural injections provide short- and long-term relief for lumbar disc herniation? A systematic review. Clin Orthop Relat Res. 2015; 473(6): 1940–56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPandey RA: Efficacy of Epidural Steroid Injection in Management of Lumbar Prolapsed Intervertebral Disc: A Comparison of Caudal, Transforaminal and Interlaminar Routes. J Clin Diagn Res. 2016; 10(7): RC05–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu J, Zhou H, Lu L, et al.: The Effectiveness of Transforaminal Versus Caudal Routes for Epidural Steroid Injections in Managing Lumbosacral Radicular Pain: A Systematic Review and Meta-Analysis. Medicine (Baltimore). 2016; 95(18): e3373. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPloumis A, Christodoulou P, Wood KB, et al.: Caudal vs transforaminal epidural steroid injections as short-term (6 months) pain relief in lumbar spinal stenosis patients with sciatica. Pain Med. 2014; 15(3): 379–85. PubMed Abstract \| Publisher Full Text\n\nPark JW, Nam HS, Cho SK, et al.: Kambin's Triangle Approach of Lumbar Transforaminal Epidural Injection with Spinal Stenosis. Ann Rehabil Med. 2011; 35(6): 833–43. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee JH, Shin KH, Bahk SJ, et al.: Comparison of clinical efficacy of transforaminal and caudal epidural steroid injection in lumbar and lumbosacral disc herniation: A systematic review and meta-analysis. Spine J. 2018; 18(12): 2343–2353. PubMed Abstract \| Publisher Full Text\n\nKircelli A, Cansever T, Yılmaz C: The influence of adjunctive caudal epidural steroid injection on the therapeutic effect of transforaminal epidural steroid injection. Neurol India. 2018; 66(1): 90–5. PubMed Abstract \| Publisher Full Text\n\nChun EH, Park HS: Effect of High-Volume Injectate in Lumbar Transforaminal Epidural Steroid Injections: A Randomized, Active Control Trial. Pain Physician. 2015; 18(6): 519–25. PubMed Abstract\n\nSanjaroensuttikul N: The Oswestry low back pain disability questionnaire (version 1.0) Thai version. J Med Assoc Thai. 2007; 90(7): 1417–22. PubMed Abstract\n\nSithapan M: data_epidural_04042020.xls. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12320846.v2\n\nRydevik B, Brown MD, Lundborg G: Pathoanatomy and pathophysiology of nerve root compression. Spine (Phila Pa 1976). 1984; 9(1): 7–1.5. PubMed Abstract \| Publisher Full Text\n\nOlmarker K, Redevik B, Holm S: Edema formation in spinal nerve roots induced by experimental, graded compression. An experimental study on the pig cauda equina with special reference to diff erences in effects between rapid and slow onset of compression. Spine (Phila Pa 1976). 1989; 14(6): 569–73. PubMed Abstract\n\nLee IS, Kim SH, Lee JW, et al.: Comparison of the temporary diagnostic relief of transforaminal epidural steroid injection approaches: conventional versus posterolateral technique. AJNR Am J Neuroradiol. 2007; 28(2): 204–8. PubMed Abstract\n\nRabinovitch DL, Peliowski A, Furlan AD: Influence of lumbar epidural injection volume on pain relief for radicular leg pain and/or low back pain. Spine J. 2009; 9(6): 509–17. PubMed Abstract \| Publisher Full Text\n\nLee F, Jamison DE, Hurley RW, et al.: Epidural lysis of adhesions. Korean J Pain. 2014; 27(1): 3–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDesai MJ, Shah B, Sayal PK: Epidural contrast flow patterns of transforaminal epidural steroid injections stratified by commonly used final needle-tip position. Pain Med. 2011; 12(6): 864–70. PubMed Abstract \| Publisher Full Text\n\nFurman MB, Butler SP, Kim RE, et al.: Injectate volumes needed to reach specific landmarks in S1 transforaminal epidural injections. Pain Med. 2012; 13(10): 1265–74. PubMed Abstract \| Publisher Full Text\n\nPark DY, Kang S, Park JH: Factors Predicting Favorable Short-Term Response to Transforaminal Epidural Steroid Injections for Lumbosacral Radiculopathy. Medicina (Kaunas). 2019; 55(5): E162. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFurman MB, Mehta AR, Kim RE, et al.: Injectate volumes needed to reach specific landmarks in lumbar transforaminal epidural injections. PM R. 2010; 2(7): 625–35. PubMed Abstract \| Publisher Full Text\n\nFurman MB, Lee TS, Mehta A, et al.: Contrast flow selectivity during transforaminal lumbosacral epidural steroid injections. Pain Physician. 2008; 11(6): 855–61. PubMed Abstract\n\nOwlia MB, Salimzadeh A, Alishiri G, et al.: Comparison of two doses of corticosteroid in epidural steroid injection for lumbar radicular pain. Singapore Med J. 2007; 48(3): 241–5. PubMed Abstract\n\nKang SS, Hwang BM, Son HJ, et al.: The dosages of corticosteroid in transforaminal epidural steroid injections for lumbar radicular pain due to a herniated disc. Pain Physician. 2011; 14(4): 361–70. PubMed Abstract\n\nGrubb SA, Lipscomb HJ, Coonrad RW: Degenerative adult onset scoliosis. Spine (Phila Pa 1976). 1988; 13(3): 241–5. PubMed Abstract \| Publisher Full Text\n\nGrubb SA, Lipscomb HJ, Suh PB: Results of surgical treatment of painful adult scoliosis. Spine (Phila Pa 1976). 1994; 15(14): 1619–27. PubMed Abstract \| Publisher Full Text\n\nJackson RP, McManus AC: Radiographic analysis of sagittal plane alignment and balance in standing volunteers and patients with low back pain matched for age sex, and size. A prospective controlled clinical study. Spine (Phila Pa 1976). 1994; 19(14): 1611–8. PubMed Abstract \| Publisher Full Text\n\nPlastaras CT, Joshi AB: The electrodiagnostic evaluation of radiculopathy. Phys Med Rehabil Clin N Am. 2011; 22(1): 59–74. PubMed Abstract \| Publisher Full Text\n\nMondelli M, Aretini A, Arrigucci U, et al.: Clinical findings and electrodiagnostic testing in 108 consecutive cases of lumbosacral radiculopathy due to herniated disc. Neurophysiol Clin. 2013; 43(4): 205–15. PubMed Abstract \| Publisher Full Text\n\nSithapan M: Information of abbreviation data set. figshare. Dataset. 2020. http://dx.doi.org/10.6084/m9.figshare.12361499.v1\n\nSithapan M: Consort checklist. figshare. Dataset. 2020. http://dx.doi.org/10.6084/m9.figshare.12361490.v1" }	[ { "id": "65445", "date": "24 Jun 2020", "name": "Steven P. Cohen", "expertise": [ "Reviewer Expertise Pain Medicine" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have performed a small, randomized trial comparing transforaminal epidural steroid injection (TFESI) to TFESI plus add-on caudal ESI. This is an interesting concept, there are not many randomized trials that compare add-on therapy (in essence, a comparative-effectiveness study). I enjoyed reading this and feel it would be a welcome addition to the literature.\nIntroduction:\nIntroduction, para 1: Steroids also suppress ectopic discharges of injured nerves, and may enhance the washout of inflammatory cytokines. This is eloquently noted in the discussion but should be noted at the beginning of the introduction as well.\n\nThe authors use the term synergistic incorrectly. Synergistic would be 20% + 20% = 50%, but while 2 treatments may afford better results than a single one (see Gilron et al. studies for neuropathic medications), the results are usually not even additive.\n\nThere is a downside to doing a caudal + TFESI even if it works better. There are 2 different billing codes, so it may not be approved. Not only does it increase costs, but it also increases the risk (compared to just using higher volumes or doses). The authors should, therefore, provide a rationale as to why the combination should work better (the background on the literature is otherwise focused and to the point).\n\nPlease consider providing objectives in the ‘introduction’ (the authors do a good job of setting the stage for the study, but specific objectives would be helpful).\n\nMethods:\nI actually like the fact that there was no minimum pain score, as this is always subjective and when a minimum is employed, patients often end up skewed towards the cutoff (i.e. a non-normal curve).\n\nOn a similar note, I also appreciate that patients with both a herniated disc and spinal stenosis. Were there any differences in outcomes?\n\nMany patients with radicular pain have bilateral symptoms. If this were the case, how did the investigators choose what side to inject? Or were these patients excluded (if this is the case, please note it in the ‘methods’)?\n\nWhy did a nurse anesthetist assess outcomes? Was sedation used (please mention)?\n\nAlthough the authors performed a power analysis, the study is likely underpowered, as well-designed studies now enroll several hundred people (see Friedly et al. NEJM, FDA-endorsed CLEAR study) to detect a difference between an ESI and a sham injection, so comparing 2 different ESI should require even more. Yet, the authors have shown a difference.\n\nPlease consider mentioning the randomization block size.\n\nThe study had excellent retention (few lost-to-follow-ups), but please note how missing data were handled in the ‘statistical analysis’ section.\n\nResults:\nI agree with one categorical outcome being > 50% pain relief, but the IMMPACT guidelines state that 30% or more relief is ‘clinically meaningful’, so most pain studies now use that cutoff as a positive outcome. According to a review by Bicket MC et al. in Pain Pract 2017, an even lower percentage of pain relief seems to predict satisfaction in most patients. So you might consider showing the proportion of people who also obtained > 30% pain relief.\n\nDuration of pain is a really important clinical variable that can have a huge effect on outcome (Bicket et al. AQUARIUS.. Reg Anesth Pain Med 2018). Please note the duration of pain for the groups.\n\nFigures 2 and 3 are wrong because the figure legends states they are showing pain scores & ODI respectively when the y-axes state they are showing ODI and pain scores, respectively (i.e. they’re reversed).\n\nDiscussion: - in general, well-written and focused.\nThere are 2 possible reasons that warrant mention as to why a difference was shown. One is that the combination group received a higher dose of steroid and local anesthetic (if there had been a group that received a higher dose of steroids by one route as a control that would have formed another control and answered questions). However, there are half a dozen studies that compare 2 different doses of steroids and nearly all have found no difference.\nThe second reason is that the group that received both injections had greater expectations, and therefore had a higher placebo response rate.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5713", "date": "24 Jul 2020", "name": "Sithapan Munjupong", "role": "Author Response", "response": "Thank your so much Dr.Cohen for your review and comment.We already revised the manuscript in Version 2 and have changed the definition of pain relief from 50% to 30%, the conclusion was changed in the subgroup analysis.All of nurse in our clinic are nurse anaesthetists. Therefore, only one nurse anaesthetist was trained to evaluated the outcome. However, the patients had not been sedated.Furthermore, we performed other answers in the revised version.Best regardsAuthors" } ] }, { "id": "65441", "date": "06 Jul 2020", "name": "Koravee Pasutharnchat", "expertise": [ "Reviewer Expertise Anesthesiology and Pain management" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors conducted an interesting randomized study comparing transforaminal epidural steroid injection (TFESI) to TFESI combined with caudal ESI. The methodology and flow of the study were organized. The analysis was properly done and results were clear.\n\nHowever, figures 2 and 3 are wrongly placed. The legends of figures state they are average verbal numerical rating scale (VNRS) and average Oswestry Low Back Pain Disability Index (ODI) respectively, while the Y-axes show ODI and VNRS, respectively.\nThe discussion is well written. Nevertheless, I think it would be fair if the two groups received the same total dosage of steroid, although many studies did show that two different doses of steroids did not make any differences. Another limitation of such a study, is that the patients were not blinded from their study groups would be a higher placebo effect in the group that received combined injections.\n\nAdditionally, it would be beneficial if the author had studied comparing cost-effectiveness between these two study groups.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5715", "date": "24 Jul 2020", "name": "Sithapan Munjupong", "role": "Author Response", "response": "Thank you for your review and comment. We already edited as new version Best regards Authors" } ] }, { "id": "65443", "date": "08 Jul 2020", "name": "Jatuporn Eiamcharoenwit", "expertise": [ "Reviewer Expertise pain management" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe primary outcome of this study was an effective response to treatment, predefined by at least a reduced verbal numerical rating scale from baseline. If the cut-off point for the percentage of pain intensity difference was 33%, the primary outcomes may be changed. A 33% pain intensity difference is a standard of the clinically important difference in pain outcome measures.1\n\nFigure 2 and figure 3 were alternated figure legends.\n\nIs the work 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": "5714", "date": "24 Jul 2020", "name": "Sithapan Munjupong", "role": "Author Response", "response": "Thank you for your review and comment. We already edited as new version Best regards Authors" } ] }, { "id": "65442", "date": "09 Jul 2020", "name": "Abdul Hadi Mohamed", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 study is very relevant to today's practice and important for patients' wellbeing and continuous healthcare for low back pain with radiculopathy.\n\nOverall coverages and discussion are appropriate.\n\nThe main limitation for this study is the imaging that showing the flow of contrast which determine of the deposition of steroid as well as the outcome.\n\nIntroduction:\nAdequate and covering the area of research.\n\nMethodology:\nThe selection of participants are adequate.\n\nI am totally agreed with the selection of a common cause of low back pain in this study.\n\nResults:\nThe table and the analysis of the result are adequate.\n\nDiscussion:\nThe coverage of the discussion and suggestion are adequate.\n\nHowever, my concern with the selection of participants who is inexperienced without comparing the senior student may affect this study's strength.\n\nReferences:\nAdequate\n\nOthers:\nThis is a good and practical study and should be encouraged to do it multicenter.\n\nThis is a very interesting study and can give us options if we may encounter partial or temporary relief with one approach. The limitation of this study is that we can confirm that limited outcome is it due to mechanical that reduces the flow of injected since no contrast was viewed prior to injection.\nFurther reading\n\nRashmi Datta, KK Upadhyay. A Randomized Clinical Trial of Three Different Steroid Agents for Treatment of Low Backache through the Caudal Route. MJAFI 2011; 67: 25-331 McCormick Z et al. Comparison of Pain Score Reduction Using Triamcinolone vs. Betamethasone in Transforaminal Epidural Steroid Injections for Lumbosacral Radicular Pain. Am. J. Phys. Med. Rehabil. 20152 Tae Kyu Park et al. Factors associated with the outcome of transforaminal epidural steroid injections. Korean J Anesthesiol. 2008; 55(3): 298-304.3\n\nIs the work 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": "5716", "date": "24 Jul 2020", "name": "Sithapan Munjupong", "role": "Author Response", "response": "Thank you for your review and comment. We already edited and add more contrast flow picture as new version Best regards Authors" } ] } ]	1	https://f1000research.com/articles/9-634
https://f1000research.com/articles/9-767/v1	24 Jul 20	{ "type": "Research Article", "title": "Isolation and characterization of a novel Sphingobium yanoikuyae strain variant that uses biohazardous saturated hydrocarbons and aromatic compounds as sole carbon sources", "authors": [ "Mautusi Mitra", "Kevin Manoap-Anh-Khoa Nguyen", "Taylor Wayland Box", "Jesse Scott Gilpin", "Seth Ryan Hamby", "Taylor Lynne Berry", "Erin Harper Duckett", "Kevin Manoap-Anh-Khoa Nguyen", "Taylor Wayland Box", "Jesse Scott Gilpin", "Seth Ryan Hamby", "Taylor Lynne Berry", "Erin Harper Duckett" ], "abstract": "Background: Green micro-alga, Chlamydomonas reinhardtii (a Chlorophyte), can be cultured in the laboratory heterotrophically or photo-heterotrophically in Tris-Phosphate-Acetate (TAP) medium, which contains acetate as the carbon source. Chlamydomonas can convert acetate in the TAP medium to glucose via the glyoxylate cycle, a pathway present in many microbes and higher plants. A novel bacterial strain, CC4533, was isolated from a contaminated TAP agar medium culture plate of a Chlamydomonas wild type strain. In this article, we present our research on the isolation, and biochemical and molecular characterizations of CC4533. Methods: We conducted several microbiological tests and spectrophotometric analyses to biochemically characterize CC4533. The 16S rRNA gene of CC4533 was partially sequenced for taxonomic identification. We monitored the growth of CC4533 on Tris-Phosphate (TP) agar medium (lacks a carbon source) containing different sugars, aromatic compounds and saturated hydrocarbons, to see if CC4533 can use these chemicals as the sole source of carbon. Results: CC4533 is a Gram-negative, non-enteric yellow pigmented, aerobic, mesophilic bacillus. It is alpha-hemolytic and oxidase-positive. CC4533 can ferment glucose, sucrose and lactose, is starch hydrolysis-negative, resistant to penicillin, polymyxin B and chloramphenicol. CC4533 is sensitive to neomycin. Preliminary spectrophotometric analyses indicate that CC4533 produces b-carotenes. NCBI-BLAST analyses of the partial 16S rRNA gene sequence of CC4533 show 99.55% DNA sequence identity to that of Sphingobium yanoikuyae strain PR86 and S. yanoikuyae strain NRB095. CC4533 can use cyclo-chloroalkanes, saturated hydrocarbons present in car motor oil, polyhydroxyalkanoate, and mono- and poly-cyclic aromatic compounds, as sole carbon sources for growth. Conclusions: Taxonomically, CC4533 is very closely related to the alpha-proteobacterium S. yanoikuyae, whose genome has been sequenced. Future research is needed to probe the potential of CC4533 for environmental bioremediation. Whole genome sequencing of CC4533 will confirm if it is a novel strain of S. yanoikuyae or a new Sphingobium species.", "keywords": [ "Chlamydomonas", "Sphingobium yanoikuyae", "TAP medium", "CC4533", "bioremediation", "16S rRNA gene", "neomycin-sensitivity", "beta-carotene" ], "content": "Introduction\n\nThe bacterium Sphingomonas belongs to the class Alphaproteobacteria, order Sphingomonadales and class Sphingomonadaceae. The genus Sphingomonas was first described by Yabuuchi et al. (1990) as a strictly aerobic, chemoheterotrophic, yellow-pigmented, Gram-negative, rod-shaped bacterium containing glycosphingolipids in cell-envelope1. Takeuchi et al. (1994) described four phylogenetic clusters in the genus based on 16S rRNA gene sequence data, and later, combined phylogenetic, chemotaxonomic and physiological analyses to split the genus into four genera: Sphingomonas, Sphingobium, Novosphingobium and Sphingopyxis2,3. Recently, the genus Sphingosinicella has been added to the family Sphingomonadaceae4. The type species of the ever-expanding genus Sphingobium is Sphingobium yanoikuyae and species with valid published names are S. amiense, S. aromaticiconvertens, S. chlorophenolicum, S. chungbukense, S. cloacae, S. francense, S. fuliginis, S. herbicidovorans, S. indicum, S. japonicum, S. olei, S. xenophagum, S. yanoikuyae, S. ummariense and S. rhizovicinum5–12.\n\nIn our research laboratory, we employ the green micro-alga Chlamydomonas reinhardtii, a member of the family Chlorophyceae, as a model system to study oxygenic photosynthesis. C. reinhardtii is grown at the laboratory in the presence (photo-heterotrophically) or absence of light (heterotrophically) in the acetate-containing Tris-Phosphate-Acetate (TAP) medium13. TAP contains 0.1% acetate as the sole carbon source13. The acetate in TAP medium is used by Chlamydomonas for net biosynthesis of glucose via the glyoxylate/C2 cycle14. This allows Chlamydomonas to make sugar heterotrophically in the dark or photo-heterotrophically in the light, without being completely dependent on photosynthesis for glucose biosynthesis14. Many bacteria possess glyoxylate cycle and can utilize acetate as a carbon source, like Chlamydomonas15,16. Hence, we often encounter bacterial contamination on Chlamydomonas TAP-agar medium culture plates. We observed a yellow-pigmented bacterial contamination on the TAP-agar medium culture plate of the Chlamydomonas wild type strain, CC4533. This bacterium was able to grow on the TAP-agar medium because it was able to use acetate as the carbon source. We named this bacterium as CC4533 because it contaminated the Chlamydomonas wild type strain CC4533. A team of four undergraduate research students from the University of West Georgia and, one high school student from the Carrollton High School in Georgia (USA) were assigned a research project to test four antibiotics that were available in our laboratory to determine which one of these four antibiotics would be suitable for use to minimize Chlamydomonas contamination by CC4533. After the antibiotic-sensitivity of CC4533 was determined, we performed additional microbiological and molecular experiments to better characterize the bacterial strain, CC4533.\n\nWe employed a modified version of the Kirby-Bauer (KB) disc diffusion antibiotic susceptibility test to determine antibiotic sensitivity of CC4533. Two different doses of four antibiotics (penicillin, neomycin, chloramphenicol and polymyxin B) were tested in the KB experiment. The results from the KB test show that CC4533 is resistant to penicillin and chloramphenicol but is sensitive to neomycin. Although CC4533 is more sensitive to polymyxin B at higher dose, polymyxin B cannot be used for controlling Chlamydomonas contamination, as at higher dose Chlamydomonas growth is affected. We found that 50 µg/mL of neomycin in TAP medium was potent for eradicating CC4533 contamination on Chlamydomonas media plates in our research laboratory, without hindering Chlamydomonas growth.\n\nWe performed standard microbiological tests to biochemically characterize CC4533. These tests show that CC4533 is an aerobic, non-enteric, Gram-negative long rod shaped bacterium (bacillus). CC4533 does not grow on Mannitol Salt Agar and MacConkey agar. CC4533 is starch hydrolysis-negative, alpha-hemolytic and, is cytochrome c oxidase-positive. CC4533 has a bright yellow pigmentation on Lysogeny Broth (LB) agar and a pale yellow pigmentation on TAP agar. CC4533 grows best at temperatures ranging from 22°C-30°C. CC4533 cannot grow at 37°C on LB agar or on TAP agar medium. Preliminary spectrophotometric analyses of extracted yellow pigment of CC4533 strongly indicate that CC4533 produces β-carotenes. Preliminary results indicate growth medium type (LB Vs. TAP) and, temperature affects β-carotene production in CC4533.\n\n0.1% acetate in the TAP medium can be substituted with alternative carbon sources (e.g. different sugars) to test if these alternative carbon sources can be used by bacteria for energy production. We monitored the ability of CC4533 to grow on Tris-Phosphate (TP) agar medium (lacks acetate) containing monosaccharide (glucose) and two disaccharides (sucrose and lactose). CC4533 was able to use these three sugars as the sole carbon source and was able to ferment these sugars to produce acid.\n\nTo determine the taxonomic identity of CC4533, we amplified the 16S rRNA gene partially by DNA polymerase Chain Reaction (PCR). We have submitted this partial 16S rRNA gene sequence to the NCBI GenBank in November 2019, with the definition: Sphingobium yanoikuyae strain PR86 variant; 16S ribosomal RNA gene, partial sequence (Accession number: MN633285.1). Our definition was based on the nearest relative identified by the NCBI-nucleotide BLAST analyses of the partial 16S rRNA gene sequence of CC4533 in 2019, which was Sphingobium yanoikuyae strain PR86 (Accession number: MN232173.1). In April 2020, we found another close relative of CC4533 in the NCBI database based on the partial 16S rRNA gene sequence, which was not detected in our earlier BLAST analyses in 2019. This new relative is Sphingobium yanoikuyae strain NRB095 (Accession number: MK543001.1). 16S rRNA partial gene sequences of these two Sphingobium yanoikuyae strains have identical: score, percentage of sequence identity, E-values and nucleotide changes in the C4 region at identical locations in the 16S rRNA gene, relative to that of CC4533.\n\nSphingomonas sp. and Sphingobium sp. are of particular interest due to their abilities to degrade cycloalkanes and polycyclic aromatic hydrocarbons (PAH) and polyhydroxyalkanoates (PHA) and their roles in environmental bioremediation17–40. Hence, we monitored the growth of CC4533 on TP-agar medium plates containing common biohazardous chemicals that are found in our natural environment. We tested the following chemicals: saturated hydrocarbons, cyclohexyl chloride, aromatic acid like benzoate, aromatic ester like phenyl acetate, polyesters (polyhydroxybutyrate, a biodegradable plastic) and poly-cyclic aromatic hydrocarbons. Growth analyses revealed that CC4533 is capable of utilizing these toxic organic compounds as the sole carbon and energy source. In future, we plan to sequence the whole genome of CC4533. This will provide important insights into the metabolic diversity of CC4533 and, will reveal the genes that are recruited by this bacterium to generate biochemical pathways for degradation of xenobiotics. In this article, we present our student-driven research on the isolation of CC4533 (Sphingobium yanoikuyae strain PR86 variant) and its characterizations at the biochemical and molecular level.\n\n\nMethods\n\nChlamydomonas wild type strain 4A+ (CC- 4051 4A+ mt+) was maintained in the lab on TAP agar medium in dim light intensities (15-20 µmol m-2s-1) at 22°C (room temperature). Standard TAP medium recipe can be found at the website of Chlamydomonas Resource Center. Hutner’s trace element solution is an ingredient in the TAP medium. Hutner’s trace element recipe can also be found on the Chlamydomonas Resource Center website. Our lab’s TAP medium recipe is slightly different from the standard one and can be found at https://doi.org/10.17504/protocols.io.bgzujx6w41. Liquid 4A+ cultures were grown in liquid TAP medium under low light (50-60 µmol m-2s-1) for 3 days on a New Brunswick Scientific Excella E5 platform shaker (Enfield, CT) at 150 rpm for aeration. CC4533 bacterial strain stock was maintained in the lab under dim light (15-20 µmol m-2s-1) at 22°C on either TAP or LB agar medium. Liquid cultures of CC4533 were grown in culture tubes in 3 mL of TAP medium at 22°C on a MaxQ420HP incubator shaker (Thermo Fisher Scientific, Waltham, MA) at 200 rpm for aeration. Light intensities were measured using a LI-250A light meter (LI-COR, Inc., Lincoln, NE).\n\nImages of all media plates used in the experiments were imaged with a Samsung Galaxy S5 cell phone camera. Image cropping and adjustments were made using Photos app in Windows 10. DNA gels were visualized and imaged with a Bio-Rad Molecular Imager Gel Doc XR+ (Bio-Rad, Hercules, CA).\n\nAntibiotics that were tested are: penicillin; neomycin, chloramphenicol and polymyxin B. Two doses (50 µg and 100 µg) of each of these four antibiotics were tested in the modified Kirby-Bauer (KB) disc diffusion antibiotic susceptibility tests. KB tests were performed on TAP-agar plates as described in Mitra et al. 202041–43. Antibiotic plates were incubated at 22°C. CC4533 plates were imaged after 3 days of incubation and Chlamydomonas plates were imaged after 4 days of incubation. Diameters of zone of inhibitions were measured using a ruler. Statistical analyses44 and images of all antibiotic plates45 are available as Underlying data.\n\nStandard deviations and means of the zones of inhibitions from the KB test were calculated using Microsoft Excel. Statistical analyses of the data from the KB disc diffusion test were performed using Microsoft Excels’ t-Test: Paired Two Sample for Means tool in the analysis ToolPak.\n\n1) Growth at different temperatures: CC4533 was streaked on fresh LB and TAP agar medium. Streaked CC4533 media plates were incubated at different temperatures: a) 22°C, b) 30°C, and c) 37°C, for 5 days and then imaged.\n\n2) Growth assays on MacConkey agar (MAC) and Mannitol Salt Agar (MSA): CC4533 was streaked on MAC and MSA plates purchased from Carolina Biological (Burlington, NC). Streaked plates were incubated at 30°C for 3 days. After the incubation period, plates were imaged to monitor CC4533 growth and pH change in the media.\n\n3) Testing CC4533’s ability to secrete hemolysins: Growth was monitored at 30°C on tryptic soy agar medium plates containing 5% sheep agar (Carolina Biological; Burlington, NC) for a period of 72 hours. Images were taken after every 24 hours over a period of three days. Classification of hemolysis were assigned according to https://www.asm.org/Protocols/Blood-Agar-Plates-and-Hemolysis-Protocols. Images of all tryptic soy blood agar plates are available as Underlying data46.\n\n4) Growth assays of CC4533 on Tris-Phosphate (TP)-phenol red-sugar- agar medium: TP medium has all the ingredients of the TAP medium except the acetate (https://doi.org/10.17504/protocols.io.bgzujx6w)41. CC4533 was streaked on three types of TP-phenol red-agar medium containing 1% glucose or 1% sucrose or 1% lactose. pH of the TP medium was 7.2. TP-sugar medium plates were imaged after 5 days of growth at 22°C. Results were interpreted as described in Mitra et al. 202041.\n\n5) Growth assays of CC4533 on TP agar media plates containing saturated hydrocarbons and aromatic compounds: Growth assays were performed on TP-agar plates coated with different doses of cyclohexyl chloride, polyhydroxybutyrate, phenanthrene, naphthalene, benzoic acid, phenyl acetate and fresh and combusted 10W30 oil, using a modified technique as described in Mitra et al. 202041,47. CC4533 was streaked on the chemical-coated TP plates and incubated at 22°C and media plates were imaged after two weeks. Images of all media plates are available as Underlying data48.\n\n6) Gram Staining, cytochrome c oxidase test and starch hydrolysis test: Gram staining was performed using CC4533 cells from LB-agar medium plate, as described in Mitra et al. 202041. Oxidase test was performed using CC4533 and a Microbacterium sp. cells from tryptic soy agar medium plates as described (with explanations) in Mitra et al. 2020 and ASM, Protocols (https://www.asm.org/Protocols/Oxidase-Test-Protocol; https://microbeonline.com/oxidase-test-principle-procedure-and-oxidase-positive-organisms/)41. For starch hydrolysis test, CC4533 and Escherichia coli were streaked on Mueller-Hinton agar medium plates purchased from Carolina Biological (Burlington, NC) and incubated for 48 hours at 30°C. Starch hydrolysis test was performed as described (with explanations) in Mitra et al. 202041.\n\nLB-grown CC4533 cells and mashed baby carrots were used for pigment extraction. Pigments were extracted with 4 mL of 100% acetone by incubation in dark for 3.5 hours at room temperature. After incubation, the sample tubes were vortexed and then centrifuged at X 4000g for 5 minutes. The yellow supernatant from CC4533 and carrot sample tubes were collected and the cell pellet/tissue debris were discarded. The yellow supernatant was then filtered using a 5 mL syringe fitted to a nylon membrane filters with a cut-off of 0.45 µm. Pigments were analyzed by direct absorbance measurements using the wavelength scan program ranging from 400-600 nm in a Beckman Coulter DU 730 Life science UV/Vis spectrophotometer (Brea, CA). The carrot supernatant absorbance scan was used as a reference overlay against the CC4533 measured scan. To detect carotenoids in the samples, absorption peaks in the 420-490 nm region were monitored (https://assets.publishing.service.gov.uk/media/57a08cbae5274a31e00013d4/tech02.pdf)49,50.\n\nGenomic DNA was isolated from CC4533 using Qiagen’s blood and cell culture DNA mini kit (Qiagen, Valencia, CA) according to the protocol given in the technical manual. Purity of the isolated genomic DNA and its concentration were measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Genomic DNA quality was determined by visualization of the genomic DNA after it was separated by DNA agarose gel electrophoresis.\n\n16S rRNA gene forward (16SF) and reverse (16SR) PCR primers were designed based on primer sequences given in article by Klindworth et al. (2013)51. Primer sequences, details about PCR cycling conditions, specific DNA polymerase used in the PCR, separation and visualization of PCR samples can be found in the article by Mitra et al. 202041.\n\nThe partial 16S rRNA genomic PCR product was extracted and purified from the DNA agarose gel using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). The purified PCR product was cloned as described in Mitra et al. 202041. One clone was sequenced using Sanger Dideoxy sequencing at the UC Berkeley DNA Sequencing Facility. Chromas Lite and nucleotide BLAST program were used to analyze the partial 16S rRNA gene sequences. Raw electropherogram files and DNA sequence text files are available as Underlying data52.\n\n\nResults\n\n\n\n\n\n\n\n\n\n\n\nWe found a yellow pigmented-bacterial contamination on a TAP medium plate of a Chlamydomonas wild type strain, CC4533, at our laboratory (Figure 1A). We purified the bacterial strain from the contaminated TAP-agar medium plate by picking single colonies on fresh TAP agar medium. We picked 40 single colonies and transferred these colonies to fresh LB agar medium. Colony # 28 was selected for our studies (Figure 1B). Colony # 28 stock was maintained in the lab on LB agar medium at 22°C (Figure 1C). We named this bacterial strain as CC4533, as the bacterium was isolated from the Chlamydomonas strain, CC4533 TAP-agar medium plate. CC4533 bacterial strain can use acetate as a carbon/energy source. Hence it was able to grow on the TAP-agar medium (Figure 1A).\n\n(A) Tris-Acetate-Phosphate (TAP)-agar medium plate showing bacterial contamination of a Chlamydomonas strain, CC4533 at room temperature (22°C). (B) A LB (Lysogeny Broth)-agar medium plate showing some of the single colonies of CC4533 that were picked. Colony # 28 outlined by the black circle was selected for culture stock maintenance and further analyses. (C) LB-agar medium plate of purified CC4533 strain. Culture plate shown in (C) was imaged after 5 days of growth at 22°C.\n\nWe performed two different sets of experiments for testing antibiotic-sensitivity. In the first experiment, we determined the antibiotic-sensitivity of CC4533 and Chlamydomonas 4A+ wild type strain to identify a suitable antibiotic and the required dose that would inhibit growth of CC4533 but will not affect the growth of Chlamydomonas. In the second set of experiments, we streaked Chlamydomonas and bacterium CC4533 together on the TAP-agar medium plate containing the antibiotic at the proper dose that we determined based on the results obtained from the first experiment set. We used two different doses (50 µg and 100 µg) of each of the following four antibiotics: penicillin, chloramphenicol, neomycin and polymyxin B. Mean diameter of the zone of inhibition for each antibiotic dose with the standard deviations are shown in Table 1. Detailed statistical analyses of the data from three biological replicates (each of which had three internal replicates) are available as Underlying data44. Images of TAP antibiotic-agar plates from the KB experiments are available as Underlying data45.\n\nZones of inhibitions induced by four different antibiotics were studied for Chlamydomonas reinhardtii and the bacterial strain, CC4533. Grey and white rows represent 50 µg and 100 µg dose of each antibiotics applied on the filter paper discs, respectably. Three biological replicates with three internal replicates were used to calculate the mean and standard deviations shown in the table. Statistical analyses44 and images of all antibiotic plates45 are available as Underlying data.\n\nAt 50 µg dose, bacterium CC4533 was more sensitive to polymyxin B than Chlamydomonas and, both Chlamydomonas and CC4533 were sensitive to the 100 µg dose of polymyxin B (Table 1; Underlying data44,45). Statistical analyses supported that the 50 µg dose of polymyxin B is not very effective in inhibiting growth of CC4533, without drastically affecting Chlamydomonas growth (Underlying data44,45). Both Chlamydomonas and CC4533 were sensitive to 100 µg (1000 units) dose of polymyxin B, but there was a significant statistical difference between the polymyxin B-sensitivity of Chlamydomonas and CC4533 at the 100 µg dose (Underlying data44,45; Table 1). Both CC4533 and Chlamydomonas were resistant to the two doses of penicillin namely, 50 µg (82.5 IU units) and 100 µg (165 IU units) as no zone of inhibition was visible for both doses (Underlying data44,45; Table 1).\n\nChlamydomonas and CC4533 were both resistant to the 50 µg and 100 µg of chloramphenicol as no zone of inhibition was obtained in the KB test (Underlying data44,45; Table 1). Chlamydomonas was less sensitive to both 50 µg and 100 µg dose of neomycin than bacterium CC4533 (Underlying data44,45; Table 1) and the sensitivity was statistically highly significant (Underlying data44,45; Table 1). Taken together, our KB test results showed that neomycin would be the best antibiotic choice to eliminate CC4533 contamination of Chlamydomonas. Polymyxin B at a higher dose (see results) could be the second choice of antibiotic for minimizing CC4533 contamination, but Chlamydomonas growth will be also affected at the higher dose of polymyxin B.\n\nIn the second experiment, we tested combined growth of CC4533 and the wild type Chlamydomonas strain 4A+ on TAP agar plates containing 50 µg and 100 µg of polymyxin B and neomycin per mL of the TAP medium (Figure 2). CC4533 was able to grow along with Chlamydomonas on TAP media plates containing 50 µg and 100 µg of polymyxin B per mL of the TAP medium (Figure 2A and Figure 2B). CC4533 did not grow on TAP plates containing 50 µg and 100 µg of neomycin per mL of the TAP medium (Figure 2C and Figure 2D). Chlamydomonas grew slowly on the TAP medium plate containing 100 µg neomycin/mL of TAP but grew at a normal rate on the TAP medium plate containing 50 µg neomycin/mL of TAP (Figure 2C and Figure 2D). Hence neomycin at a concentration of 50 µg/mL in the TAP medium is most effective in inhibiting the growth of bacterium CC4533 on Chlamydomonas culture plates without affecting the growth of Chlamydomonas.\n\n(A) Chlamydomonas strain 4A+ and CC4533 strain streaked on TAP-agar plate containing 50 µg of polymyxin B/mL of medium. (B) Chlamydomonas strain 4A+ and CC4533 strain streaked on TAP-agar plate containing 100 µg polymyxin B/mL of medium. (C) Chlamydomonas strain 4A+ and CC4533 strain TAP-agar plate containing 50 µg of neomycin/mL of medium. (D) Chlamydomonas strain 4A+ and CC4533 strain streaked on TAP-agar plate containing 100 µg neomycin/mL of medium. TAP-agar antibiotic plates were incubated at room temperature for 2.5 weeks before they were imaged.\n\nGram staining of CC4533 revealed that CC4533 is a Gram-negative bacillus. Cells are straight long rods which join to form chains (Figure 3).\n\nCC4533 cells from a LB-agar medium plate was used for gram-staining. Gram-stained cells were visualized and imaged under an oil immersion lens of a bright-field microscope.\n\nWe have monitored the growth of CC4533 on LB-agar and TAP-agar over 5 days at different temperatures namely 22°C, 30°C and 37°C. CC4533 grew well on LB and TAP-agar at 22°C and 30°C but could not grow at 37°C (Figure 4). Yellow pigmentation of CC4533 was visibly reduced on TAP medium compared to that on the LB medium. Growth at 30°C reduced pigment accumulation compared to that at 22°C on both LB and TAP medium (Figure 4).\n\n(A) Growth on LB-agar medium plate at room temperature (22°C). (B) Growth on TAP-agar medium plate at 22°C. (C) Growth on LB-agar medium plate at 30°C. (D) Growth on TAP-agar medium plate at 30°C. (E) Growth on LB-agar medium plate at 37°C. (F) Growth on TAP-agar medium plate at 37°C. Culture plates were imaged after 5 days of growth.\n\nWe grew CC4533 on TP (lacks the carbon source, acetate) agar medium containing three different types of sugars (glucose, sucrose and lactose) and the pH indicator, phenol red (Figure 5). Figure 5A, Figure 5C and Figure 5E represent control TP + 1% glucose, TP + 1% sucrose and TP + 1% lactose, plates, respectively. CC4533 grew very well on all sugar supplemented TP agar plates (Figure 5). It fermented sugars on all TP-sugar medium plates to produce acid which lowered the pH in the TP medium. The drop in pH, changed the color of phenol red from light red color to a yellow color (Figure 5B, Figure 5D and Figure 5F).\n\n(A) Control TP + 1% glucose agar medium plate with phenol red as a pH indicator. (B) CC4533 on TP +1% glucose agar medium plate with phenol red as a pH indicator. (C) Control TP +1% sucrose agar medium plate with phenol red as a pH indicator. (D) CC4533 growth on TP +1% sucrose agar medium plate with phenol red as a pH indicator. (E) Control TP +1% lactose agar medium plate with phenol red as a pH indicator. (F) CC4533 growth on TP +1% lactose agar medium plate with phenol red as a pH indicator. Culture plates were imaged after 5 days of growth at room temperature. CC4533 fermented glucose, sucrose and lactose as evident from the yellow color of phenol red.\n\nCC4533 was unable to grow on MAC and E. coli, a Gram-negative enteric bacterium (Figure 6A), was able to grow on MAC. MAC contains lactose as the carbon source. The pink color of E. coli on MAC plate, indicated that it can ferment the sugar lactose present in the MAC medium to produce acid as the acidic pH (pH below 6.8) changed the color of neutral red to pink (Figure 6B). The drop in the pH in the MAC medium around the growth of E. coli, precipitated the bile salts out of the MAC medium which caused a hazy pink zone to develop around the E. coli growth (Figure 6B). CC4533 can use lactose as a carbon source (Figure 5F). CC4533 cannot grow on MAC because it is sensitive to bile salts and crystal violet in the MAC medium (Figure 6A). Our results indicate that CC4533 is a non-enteric bacterium unlike E. coli.\n\n(A) 72 hours-growth of CC4533 on MacConkey Agar medium plate. (B) 72 hours growth of Escherichia coli on MacConkey Agar medium plate. CC4533 fails to grow on MacConkey agar medium plate. E. coli appears pinkish because it ferments lactose to acid, which causes the neutral red pH indicator to turn red. The dark opaque pink haze on the medium around the E. coli growth is the bile precipitation in acidic environment. Media plates were incubated at 30°C.\n\nCC4533 fails to grow on MSA (Figure 7A). Staphylococcus aureus grew on MSA and fermented the sugar-alcohol, mannitol, present in the MSA medium to produce acid, which changed the phenol red’s color from red to yellow (Figure 7B). Our results show that CC4533 is salt-sensitive as MSA contains about 7.5-10% of NaCl, which inhibits growth of many gram-negative bacteria and, allows selection of high salt-tolerant gram-positive bacterium like Staphylococcus.\n\n(A) 72 hours-growth of CC4533 on Mannitol Salt Agar medium plate. (B) 72 hours growth of Staphylococcus aureus on Mannitol Salt Agar medium plate. CC4533 fails to grow on Mannitol Salt Agar medium plate. S. aureus grows on Mannitol Salt Agar and can ferment mannitol. Media plates were incubated at 30°C.\n\nCC4533 did not show any hemolysis or discoloration of blood agar medium after 24 hours growth (Underlying data46). After 48 hours, a dark brown discoloration around the cell growth was observed (Underlying data46), which became more pronounced after 72 hours of growth (Figure 8A), indicating CC4533 is alpha-hemolytic. Figure 8B shows that S. aureus is beta-hemolytic as complete hemolysis can be seen around the S. aureus growth after 24 hours of growth. After 72 hours, the clearing on the blood agar medium plate is more pronounced. Images of tryptic soy blood agar plates are available as Underlying data46 and in Figure 8B.\n\n(A) 72-hours growth of CC4533 on tryptic soy-blood agar plate. (B) 72 hours growth of Staphylococcus aureus on tryptic soy-blood agar plate. Media plates were incubated at 30°C. Images of 24 hours- and 48-hours growth of both strains are available as Underlying data46.\n\nWe performed starch hydrolysis test on CC4533 and E. coli on Mueller-Hinton agar medium, which contains 0.15% starch (Figure 9). Background and scientific basis of the starch hydrolysis test can be found in Mitra et al. 202041. Both CC4533 (Figure 9B) and E. coli (Figure 9D) cannot hydrolyze starch as there was no visible clear zone around the growth on the Mueller Hinton agar. Iodine used in the starch hydrolysis test reacted with the starch present in Mueller-Hinton media to produce a brown/blue color. The results show that both bacterial strains do not secrete the enzymes α-amylase and oligo-1, 6-glucosidase to hydrolyze amylose and amylopectin (starch). We did not have a starch hydrolysis-positive strain in our lab to use as a positive control in this experiment.\n\n(A) 48 hours-growth of CC4533 at 30°C on Mueller-Hinton medium which contains 0.15% starch. (B) CC4533 Mueller-Hinton plate shown in (A) was treated with Gram iodine. (C) 48 hours-growth of E. coli at 30°C on Mueller-Hinton medium which contains 0.15% starch. (D) E. coli Mueller-Hinton plate shown in (C) treated with Gram iodine. E. coli and CC4533 fail to hydrolyze starch on Mueller-Hinton medium as there are no visible clear zones around the bacterial growth after gram iodine treatment. The brown color of the medium upon Gram iodine treatment occurs because of the reaction of starch in the medium with iodine.\n\nAerobic, facultative anaerobic or microaerophilic bacteria that uses cytochrome c oxidase in the electron transport chain associated with cellular respiration can be identified by the oxidase test. We conducted the oxidase test on CC4533 and on a yellow pigmented-Microbacterium sp., using a disposable slide that contains a film coated with oxidase reagent tetramethyl-p-phenylenediamine (TMPD). CC4533 oxidized the TMPD to indophenols, a purple colored product, within 5–10 seconds. CC4533 is oxidase-positive (Figure 10; left). Microbacterium sp. is oxidase-negative as it failed to change the color of TMPD to purple within 5–10 seconds. (Figure 10; right). We grow CC4533 under aerobic conditions in our lab (Figure 1–Figure 9). Our results show that CC4533 is an aerobic bacterium that uses cytochrome c in the respiratory electron transport chain.\n\nCells of CC4533 (on the left) and Microbacterium sp. (on the right) streaked on a disposable slide containing a film coated with oxidase reagent (tetramethyl-p-phenylenediamine dihydrochloride). Image of the slide was taken after 10 seconds of the application of the cells on the slide. CC4533 is cytochrome c oxidase-positive as cytochrome c oxidase, if present, oxidizes the oxidase reagent on the film to form purple colored-indophenols. Microbacterium sp., a yellow-pigmented bacterium, is oxidase-negative and fails to form the purple-colored product within 10 seconds.\n\nIt is known that many bacteria accumulate carotenoids, which gives them orange to yellow pigmentation. As CC4533 is yellow-pigmented, we tested for the presence of carotenoids in CC4533, grown under normal room light (30-40 µmol m-2s-1). We monitored the absorption spectrum of the acetone-extracted CC4533 pigment using wavelength scan program ranging from 400- 600 nm in a UV-Vis spectrophotometer. Carotenoids absorb strongly in the visible light range from 400-495 nm, with absorption peaking near 450 nm (https://assets.publishing.service.gov.uk/media/57a08cbae5274a31e00013d4/tech02.pdf)49,50. We found two major absorption peaks in the 400-500 nm region of the spectrum (Figure 11)49,50. The absorption peak with the absorbance reading (0.294) is at 453 nm and the one with the absorbance reading of 0.253 is at 480 nm (Figure 11B and Figure 11C). β-carotene shows two major absorption peaks that range between 451 nm - 454 nm and 477 nm - 480 nm49,50. The observed two absorption peaks in CC4533 pigment extract strongly indicates the presence of β-carotene in CC4533.\n\n(A). Absorption curve of the yellow pigment extract of CC4533. (B) Absorption peak of pigment extract at 453 nm. (C) Absorption peak of pigment extract at 480 nm. CC4533 cells were harvested for pigment extraction from a LB agar medium plate maintained under low light. Absorption maxima were measured using the wavelength scan program with the wavelength range of 400-600 nm in a UV-Vis spectrophotometer.\n\n80% of the carotenoids in carrots is β-carotenes53,54. We extracted pigments from carrots and measured its absorption peaks (Figure 12). We used the carrot pigments’ absorption curve as a reference scan overlay against the measured scan of CC4533 pigment extract (Figure 12). We found that the extracted- carrot pigments exhibited three major peaks that are representative of β-carotene absorption at 429 nm, 451 nm and 477 nm (https://assets.publishing.service.gov.uk/media/57a08cbae5274a31e00013d4/tech02.pdf; Figure 12A, Figure 12C, Figure 12D; Figure 12E). Two of these peaks (451 nm and 477 nm) are very close to that of absorption peaks (453 nm and 480 nm) of CC4533 pigment extract (Figure 11B, Figure 11C, Figure 12B, Figure 12D and Figure 12E). The 429 nm peak in the pigment extract of carrot was not prominently visible in the pigment extract of CC4533 (Figure 12C). Our preliminary data strongly indicates the presence of β-carotene in CC4533.\n\n(A) Absorption curve of extracted pigments from baby carrots (B) Absorption curve of the extracted yellow pigment from CC4533. (C) Measured scan of CC4533 pigment absorption (black plot) against the overlay of the reference scan of carrot extract (light blue plot). The absorption curve shows the carotene absorption peak at 429 nm and the corresponding difference in absorbance readings between the two samples. (D) Measured scan of CC4533 pigment absorption (black plot) against the overlay of the reference scan of carrot extract (light blue plot). The absorption curve shows the β-carotene absorption peak at 451 nm and the corresponding difference in absorbance readings between the two samples. (E) Measured scan of CC4533 pigment absorption (black plot) against the overlay of the reference scan of carrot extract (light blue plot). The absorption curve shows the β-carotene absorption peak at 477 nm and the corresponding difference in absorbance readings between the two samples. CC4533 cells from a LB agar medium plate maintained under low light was used for pigment extraction. Absorption maxima were measured using the wavelength scan program with wavelength range of 400-600 nm in a UV-Vis spectrophotometer.\n\nThe full length 16S rRNA gene is 1541 bp long (Figure 13A; based on E. coli 16S rRNA gene). There are nine hypervariable (V1-V9) and nine conserved regions (C1- C9) in the 16S rRNA gene55–57. 11 nucleotides (788-798) within the C4 conserved region are totally conserved in bacteria58. This super-conserved region is represented in Figure 13 as a black box within the C4 region58. Forward and reverse PCR primers are represented by black arrows in the schematic of the 16S rRNA gene (Figure 13A). PCR amplification of the partial 16S rRNA gene of CC4533 generated an amplicon of approximately 460 bp in size (Figure 13B). The amplicon shown in Figure 13B was sequenced to determine the nearest relative of CC4533.\n\n(A) A schematic diagram showing the conserved and hypervariable regions in the 16S rRNA gene. The interspersed conserved regions (C1–C9) are shown in gray, and the hypervariable regions (V1–V9) are depicted in white. The black box within the C4 region represents 11 nucleotides (788 -798 base pairs) that are invariant in bacteria. PCR primers are shown in thick black arrows. Forward primer is in the C2 region and the reverse primer is in the C4 region. The figure is based on the 16S rRNA gene sequence of E. coli. (B) A DNA agarose gel showing the results of PCR with the primers shown in Figure 13A. Lane 1 represents PCR with water (zero DNA control) and Lane 2 shows the PCR product from a bacterial strain LMJ41 isolated by our lab and, Lane 3 shows the CC45333 PCR product. 1kb plus DNA ladder was used as a DNA molecular size ladder on the agarose gel. (C) A schematic diagram showing the nucleotide changes in CC4533 in the 16S rRNA region spanning the C2 and C4 regions in comparison to the best NCBI- BLAST hit (score of 802; E-value 0 and percent identity of 99.55%): Sphingobium yanoikuyae strain PR86 16S ribosomal RNA gene, partial sequence (GenBank Accession #: MN232173.1). Black nucleotides show the native nucleotides in the BLAST hit Sphingobium yanoikuyae strain PR86 that were substituted by the depicted red nucleotides in CC4533 16S rRNA gene sequence. The black bold numbers within the parenthesis beside the nucleotides show the specific nucleotide position where the nucleotide changes have occurred. Nucleotide positions shown in the figures have been assigned according to that of the 16S rRNA gene sequence of E. coli. DNA sequencing data is available as Underlying data52.\n\nIn fall 2019, NCBI-nucleotide BLAST analyses identified the nearest relative of CC4533 as Sphingobium yanoikuyae strain PR86 (GenBank Accession #: MN232173.1) based on the partial 16S ribosomal RNA gene sequence. This hit has a score of 802; zero E-value and percent identity of 99.55%. Figure 13C shows two nucleotide substitutions (transitions) that are present in CC4533 16S rRNA partial gene sequence relative to that in Sphingobium yanoikuyae strain PR86. These two nucleotide substitutions are in the 11 bp super-conserved sub-region within C4 region (Figure 13C). We deposited in November 2019, the partial 16S rRNA sequence of CC4533 in GenBank with the definition: Sphingobium yanoikuyae strain PR86 variant, 16S ribosomal RNA gene, partial sequence (Accession number: MN633285.1). DNA sequencing data of the 16S rRNA gene of CC4533 are available as Underlying data52. In 2020, NCBI-BLAST analyses revealed another close relative of CC4533: Sphingobium yanoikuyae strain NRB095 (Accession number: MK543001.1) based on the partial 16S rRNA gene sequence. Both strains of Sphingobium yanoikuyae detected by our NCBI-BLAST analyses had identical scores, E-values and sequence identity (including the same nucleotide substitutions at the identical locations within the 16S rRNA gene) when their partial 16S rRNA gene sequences were compared against that of CC4533.\n\nSphingobium sp. are known to use alkanes, polycyclic aromatic hydrocarbons (PAH), polyhydroxyalkanoates like polyhydroxybutyrate (PHB) and other aromatic compounds as alternative carbon sources for growth17–40. Hence, we tested if CC4533 can utilize saturated hydrocarbons, PAH, other biohazardous aromatic compounds and PHB as carbon sources for growth. These growth analyses were performed as described in Mitra et al. 202041. Different doses of the chemicals that were tested can be also found in Mitra et al. 2020 and are also available as Underlying data48.\n\nCC4533 was streaked on a TP-agar medium plate, which lacks a carbon source, as a negative control to show that CC4533 does not grow on a TP medium plate in the absence of a carbon source (Figure 14A). CC4533 was able to grow on TP medium containing all doses of 1% cyclohexyl chloride (a mono chlorinated hydrocarbon) (Figure 14B) and 1% PHB (belongs to the class of polyhydroxyalkanoates that are used as bio-derived and biodegradable plastics) (Figure 14C). Our results show that CC4533 can use both these organic compounds as sole carbon sources for growth.\n\nTris-Phosphate (TP) agar medium plates shown in Figure 14B & Figure 14C were coated with either cyclohexyl chloride or polyhydroxybutyrate. CC4533 was streaked on the negative control TP-agar medium plate and on the hydrocarbon-coated TP-agar medium plates. After 2 weeks of growth at room temperature, media plates were imaged. (A) TP-agar medium plate streaked with CC4533. CC4533 does not grow on TP medium as it lacks a carbon source. (B) CC4533 growth on TP-agar medium plate coated with 4 mL of 1% cyclohexyl chloride diluted with chloroform. (C) CC4533 growth on TP-agar medium plate coated with 4 mL of 1% polyhydroxybutyrate.\n\nMotor oils contain petroleum-based hydrocarbons which contain between 18 and 34 carbon atoms per molecule, poly-alpha olefins or their mixtures in different ratios. We monitored the growth of CC4533 on TP medium containing 2% (v/v) fresh 10W30 car motor oil (Figure 15A–Figure B) and 2% (v/v) combusted 10W30 car motor oil (Figures 15C–D). C4533 grew on TP-agar containing all doses of 2% fresh (Figure 15A–B) and 2% combusted 10W30 motor oil (Underlying data48; Figure 15C–D), indicating that it can utilize petroleum-derived hydrocarbons as carbon sources.\n\nTris-Phosphate (TP) agar medium plates shown in Figures 15A-15D were coated with different does of fresh and combusted car motor oil. CC4533 was streaked on the motor oil-coated TP-agar media plates shown in Figures 15A-15D. After 2 weeks of growth at room temperature, media plates were imaged. (A) CC4533 growth on TP-agar medium plate coated with 0.5 mL of 2% fresh 10W-30 car motor oil. (B) CC4533 growth on TP-agar medium plate coated with 2 mL of 2% fresh 10W-30 car motor oil. (C) CC4533 growth on TP-agar medium plate coated with 0.5 mL of 2% combusted 10W-30 car motor oil. (D) CC4533 growth on TP-agar medium plate coated with 2 mL of 2% combusted 10W-30 car motor oil. TP-agar medium plate (lacks a carbon source) shown in Figure 14A served as the negative control for this experiment. Images of plates with different doses of car motor oil are available as Underlying data48.\n\nPhenanthrene is a PAH composed of three fused benzene rings. Napthalene is a PAH consisting of two fused benzene rings. CC4533 was streaked on a TP medium plate which lacks a carbon source and this plate served as the negative control in the experiment (Figure 16A). We monitored the growth of CC4533 on TP medium containing 1% phenanthrene (Figure 16B) and 1% naphthalene (Figures 16C–D). CC4533 grew on TP-agar containing all doses of 1% phenanthrene and 1% naphthalene (Underlying data48; Figures 16B–D).\n\nTP medium plates shown in Figures 16B-16D were coated with polycyclic aromatic hydrocarbons (PAH). CC4533 was streaked on the negative control TP-agar medium plate and on the PAH-coated TP-agar medium plates. After 2 weeks of growth at room temperature, media plates were imaged. (A) TP control plate streaked with CC4533. CC4533 does not grow on TP plate as it lacks a carbon source. (B) CC4533 growth on TP plate coated with 4 mL of 1% phenanthrene dissolved in chloroform. (C) CC4533 growth on TP plate coated with 0.5 mL of 1% naphthalene dissolved in chloroform. (D) CC4533 growth on TP plate coated with 2 mL of 1% naphthalene dissolved in chloroform. Images of plates with different doses of aromatic hydrocarbons are available as Underlying data48.\n\nBenzoic acid is an aromatic carboxylic acid with a single benzene ring. Benzoic acid and sodium benzoate are commonly used as food preservatives and these preservatives are major environmental pollutants. Phenyl acetate is the ester of phenol and acetyl chloride. Phenylacetate is a common environmental pollutant and is a central intermediate in the pathways that degrade aromatic chemicals (e.g. phenylalanine, phenylacetaldehyde, lignin-related phenylpropane units, environmental contaminants like styrene and ethylbenzene)59. CC4533 was able to utilize all tested doses of benzoic acid (Figures 17A–B) and phenyl acetate (Figures 17C–D) in the TP medium as the sole carbon sources (Underlying data48).\n\nTP medium plates shown in Figures 17A-17D were coated with aromatic compounds. CC4533 was streaked on the aromatic compound-coated TP-agar medium plates. After 2 weeks of growth at room temperature, media plates were imaged. (A) CC4533 growth on TP plate coated with 0.5 mL of 1% benzoate dissolved in chloroform. (B) CC4533 growth on TP plate coated with 2 mL of 1% benzoate dissolved in chloroform. (C) CC4533 growth on TP plate coated with 0.5 mL of 1% phenyl acetate dissolved in chloroform. (D) CC4533 growth on TP plate coated with 2 mL of 1% phenyl acetate dissolved in chloroform. TP-agar medium plate (lacks a carbon source) shown in (Figure 16A served as the negative control for this experiment. Images of plates with different doses of aromatic compounds are available as Underlying data48.\n\n\nDiscussion\n\nCC4533 is a mesophilic, yellow pigmented, Gram-negative rod (Figure 1–Figure 4). It can ferment glucose, sucrose and lactose (Figure 5). It is a non-enteric, salt-sensitive, alpha hemolytic, starch hydrolysis-negative, oxidase-positive bacterium (Figure 6–Figure 10). NCBI-nucleotide BLAST analyses of the partial 16S rRNA gene sequence of CC4533 revealed that the best match is to that of Sphingobium yanoikuyae strain PR86 (GenBank Accession #: MN232173.1) and to that of Sphingobium yanoikuyae strain NRB095 (Accession number: MK543001.1) with a sequence identity of 99.55% and zero E-value, indicating strongly that CC4533 is a new strain of Sphingobium yanoikuyae. Whole genome sequencing can confirm if CC4533 is a new species of Sphingobium or a new strain of Sphingobium yanoikuyae (see discussion end).\n\nThe family Sphingomonadaceae currently includes the genera Sphingomonas, Sandaracinobacter, Blastomonas, Novosphingobium, Sphingobium, Sphingopyxis, Sandarakinorhabdus, Sphingosinicella, Stakelama, Sphingomicrobium, Sphingorhabdus, Parasphingopyxis, and Zymomonas60. The type genus Sphingomonas was dissected into four genera based on clustering in the 16S rRNA gene sequence phylogeny and differences in the major polyamine and the 2-hydroxy fatty acid patterns: the genus Sphingomonas sensu stricto and the three new genera Novosphingobium, Sphingobium, and Sphingopyxis60.\n\nSphingobium sp. are chemoorganotrophs and have been isolated from soils, freshwater and marine habitats, activated sludge, phyllosphere and rhizosphere. The isolation source for Sphingobium yanoikuyae strain PR86 (GenBank Accession #: MN232173.1) is sunflower endo-phyllosphere and that for Sphingobium yanoikuyae strain NRB095 (Accession number: MK543001.1) is surface disinfested root of the tropical Koronivia grass Brachiaria humidicola. There are various examples of plant growth-promoting organisms within the Sphingomonadaceae family but the genus Sphingobium is largely limited to members that degrade xenobiotic compounds61–65. Exception is the Sphingobium sp. strain AEW4, isolated from the rhizosphere of the beach grass, Ammophila breviligulata61. This strain has plant growth promoting properties via production of siderophores and indole-3-acetic acid and induces root growth61. Sphingobium paulinellae sp. nov. and Sphingobium algicola sp. nov are isolated from Paulinella chromatophora, a freshwater filose amoeba with photosynthetic endosymbionts (chromatophores) of cyanobacterial origin66. In nature, C. reinhardtii is predominantly found in temperate, nutrient-rich, cultivated field soils in Northern America and Japan67,68. We isolated CC4533 (Sphingobium yanoikuyae strain PR86 variant) in our laboratory from TAP agar medium plates of the micro-green-alga C. reinhardtii.\n\nCC4533 is resistant to the β-lactam group of drugs like penicillin, and to the broad-spectrum antibiotic, chloramphenicol (Table 1). It is resistant to both 50 µg and 100 µg doses of the cationic antimicrobial polypeptide, polymyxin B, which is one of the most effective drugs against Gram-negative bacteria69 (Table 1; Figure 2, Underlying data44,45). CC4533 is sensitive to neomycin (Underlying data44,45; Table 1; Figure 2). The diversity and antibiotic resistance patterns of Sphingomonadaceae isolates from drinking water show that the highest antibiotic resistance prevalence values were in members of the genera Sphingomonas and Sphingobium, especially in tap water and in water from dental chairs70. Sphingobium isolates in drinking water are 89.3-100% penicillin-resistant, 96.4% polymyxin B-resistant, 25% sulfonamide-resistant and 100% neomycin-sensitive70.\n\nMany Sphingobium strains capable of degrading PAHs and other aromatic compounds have been isolated17–40. Sphingobium yanoikuyae strain B1, previously known as Sphingomonas yanoikuyae strain B1 and Beijerinckia sp. strain B1, has caught attention due to its versatile capabilities to degrade various environmental pollutants, such as biphenyl, naphthalene, phenanthrene, toluene, m-, p-xylene, anthracene17–22. Sphingobium sp. 22B isolated from soil contaminated with PAHs from Argentina has great capacity of degrading PAHs as unique sources of carbon in mineral medium and in phenanthrene microcosms assays23,24. Biphenyls and polychlorinated biphenyls (PCBs) are difficult to be completely mineralized by environmental microbes because of the accumulation of dead-end intermediates like benzoate and chlorobenzoates during biphenyl and PCB biodegradation. Sphingobium fuliginis HC3 strain isolated from PCBs-contaminated soil can degrade biphenyl and PCBs without dead-end intermediates accumulation25.\n\nSphingobium aromaticiconvertens sp. nov. isolated from polluted river sediment can mineralize monochlorinated dibenzofurans26. Sphingobium xenophagum Bayram can utilize branched 4-nonylphenol as a sole carbon source27. Sphingobium yanoikuyae strain FM-2 isolated from river water is capable of degrading Bisphenol F28. Three strains of Sphingobium sp. strain YBL1, strain YBL2 and strain YBL3 isolated from China were found to mineralize commonly used N, N-dimethyl-substituted phenylurea herbicides29,30. Sphingobium wenxiniae JZ-1 has a gene cluster that codes for phenoxybenzoic acid 1′,2′-dioxygenase, which allows this species to metabolize 3-phenoxybenzoic acid31. Sphingobium scionense sp. nov is an aromatic hydrocarbon-degrading bacterium isolated from a PAH-contaminated soil in New Zealand32. Sphingobium yanoikuyae XLDN2-5 isolated from petroleum-contaminated soils can degrade carbazole efficiently33–35. This strain could also co-metabolically catabolize dibenzofuran, dibenzothiophene, and benzothiophene33,34,36. Sphingobium yanoikuyae SHJ is a strain isolated from shallow aquifer sediment in China that can degrade diethyl phthalate, which is used as a plasticizer37.\n\nSynthesis and accumulation of PHB is widespread in bacteria and depends on either the type of strain or the carbon source used in the metabolism38,39. PHB is important for the physiology of the PAH-degrading strain Sphingobium sp. 22B, isolated from semi-arid region of Patagonia (South America) as it accumulates significant amounts of this polyhydroxyalkanoate40. PHB has been shown to play diverse roles under different degrees of water stress: it can serve as an endogenous carbon source under low water stress and can protect cells under high water stress40.\n\nWe have shown in this work that CC4533 (Sphingobium yanoikuyae strain PR86 variant) can utilize PAH, chlorinated alkanes, petroleum derivatives in 10W30 car motor oil, PHB and benzoic acids and phenyl acetate as sole carbon source in TP medium (Figure 14–Figure 17). Recently we isolated and characterized a novel bacterial strain, LMJ/Bacterium strain clone LIB091_C05_1243 variant 16S ribosomal RNA gene, partial sequence (Accession number: MN633292.1)41. The nearest relative of LMJ with a genus name is Acidovorax sp. strain A16OP12 (Accession #: MN519578.1)41. LMJ can grow on TP-medium containing saturated hydrocarbons, PHB and PAH like CC453341. LMJ hardly grows on TP medium containing phenyl acetate and benzoate unlike CC453341. CC4533 strain grows more robustly than LMJ on every tested toxic aromatic compound and hydrocarbon containing plates at all doses41. Hence, CC4533 displays a higher potential for environmental bioremediation than the bacterial strain LMJ strain41.\n\nIn future we plan to optimize the process of uniformly coating the TP medium surface with chemicals as we have noticed that the tested hydrophobic chemicals were unevenly deposited after coating, when the solvent chloroform evaporates. We think that this uneven coating can affect proper utilization of these aromatic chemicals by CC4533. Our TP medium is a nutritionally stringent minimal medium compared to the traditional M9 minimal medium used for bacterial growth41. The final concentrations of phosphate, nitrogen, magnesium and carbon in the M9 medium is approximately, 70-fold, 2.5-fold, 4-fold and 5-fold higher than that present in our lab’s TAP medium, respectively41. We would like to test if CC4533 can grow on the M9 medium without an added carbon source like a sugar or acetic acid. If CC4533 fails to grow on the M9 medium plates without a carbon source, we will compare the growth of CC4533 in M9 medium containing different PAHs or other aromatic compounds as alternative carbon sources against that in TP medium. Additionally, we need to collaborate with a research lab that can test the concentration of these toxic organic chemicals in TP medium before and after the CC4533 growth, to get additional evidence that CC4533 is degrading PAH, PHB and other hydrocarbons in the TP medium.\n\nBacterial heterocyclic aromatic compound degradation pathways mainly involve the oxidation reactions such as angular dioxygenation of carbazole and dibenzofuran, lateral dioxygenation of dibenzothiophene in Kodama pathway, and S-oxidation of dibenzothiophene in 4S pathway34,71,72. A consequence of the oxidation reaction is the formation of reactive oxygen species (ROS), which can damage DNA, proteins, and membranes73,74. Carotenoids present in a wide variety of bacteria, algae, fungi, and plants are the most prominent membrane-integrated antioxidants75. Sphingobium yanoikuyae XLDN2-5, a yellow pigmented PAH-degrading strain, synthesizes zeaxanthin from β-carotene through β-cryptoxanthin via the carotenoid biosynthetic pathway49. In Sphingobium yanoikuyae XLDN2-5, there is a direct correlation between the increase in the amount of zeaxanthin and the enhancement of hydrogen peroxide production during the biodegradation of heterocyclic aromatic compounds49. High levels of carotenoids in this Sphingobium strain were consistent with the enhanced transcription of the gene encoding phytoene desaturase, one of the key enzymes for carotenoid biosynthesis49.\n\nPigment analyses of CC4533 strongly indicate the presence of β-carotene in the CC4533 pigment extract (Figure 11; Figure 12). We plan to confirm our results by conducting HPLC analyses of the CC4533 pigment extract and a commercial pure sample of β-carotene. Carotenoid production can be measured in CC4533 grown on LB medium that contain the ROS species like hydrogen peroxide or contains photo-sensitizers like Rose Bengal that generates the ROS, singlet oxygen, in the presence of light and oxygen76. Bleaching herbicides, such as norflurazon, interferes with the carotenoid biosynthetic pathway77. Norflurazon blocks the enzyme Phytoene desaturase (PDS), which converts phytoene (colorless carotene) to red-colored lycopene78. ROS sensitivity of CC4533 can be tested in the presence and absence of the inhibitor of PDS. Carotenoids are used as regulators for membrane fluidity by Staphylococcus xylosus79. For two Staphylococcus xylosus strains there was an increase in staphyloxanthin and other carotenoids when grown at 10°C but no carotenoids could be detected when grown at 30°C79. CC4533 cannot grow at 37°C and appears lot less yellow-pigmented at 30°C compared to when it is grown at 22°C (Figure 4). Pigment reduction is more pronounced in CC4533 on TAP medium than on LB (Figure 4). Quantitative carotenoid assays at different temperatures can be performed to study the temperature effect on pigment production.\n\nPartial 16S rRNA gene sequence of CC4533 shows two transitional nucleotide changes when compared to that of the Sphingobium yanoikuyae strain PR86 (GenBank Accession #: MN232173.1) and to that of Sphingobium yanoikuyae strain NRB095 (Accession number: MK543001.1). These two nucleotide substitutions are in the 11 bp invariable sub-region (788 bp -798 bp) within C4 region (Figure 13C). These results show that conserved regions of the 16S rRNA are not truly “conserved”. Conserved regions of the 16S rRNA gene exhibit considerable variations that need to be considered when using this gene as a biomarker80.\n\nSphingomonads have attracted the attention of microbiologists and biotechnologists due to their biodegradative and biosynthetic capabilities, and have been utilized for a wide range of biotechnological applications from bioremediation of contaminants to production of extracellular polymers28. We have shown in this study that CC4533 is a Sphingobium yanoikuyae strain and has traits that can be exploited for bioremediation upon further research. We found in the NCBI database, 20 genome assemblies of Sphingobium yanoikuyae (Representative genome: Sphingobium yanoikuyae ATCC 51230; ID: 3110) and 70 genome assemblies of uncharacterized environmental isolates. Because of funding limitations, we could not sequence the whole genome of CC4533 at the time of this manuscript submission. But we will have funds in fall 2020 to sequence the whole genome of CC4533 using the Pacific Biosciences technology. Whole genome sequencing will allow us to: 1) confirm if CC4533 is a novel species of Sphingobium or a new strain of Sphingobium yanoikuyae and, 2) will reveal genes in CC4533 that are recruited for degradation of xenobiotics and contribute to its metabolic diversity, relevant to environmental bioremediation and industrial biotechnology.\n\n\nData availability\n\nSphingobium yanoikuyae strain PR86 16S ribosomal RNA gene, partial sequence. Accession number, MN633285.1: https://www.ncbi.nlm.nih.gov/nuccore/MN232173.1\n\nFigshare: Antibiotic sensitivity data of the bacterial strain CC4533 (Sphingobium yanoikuyae strain PR86 variant; GenBank Accession number: MN633285.1) and Chlamydomonas reinhardtii strain 4A+. https://doi.org/10.6084/m9.figshare.12465863.v144\n\nThis project contains the following underlying data:\n\nCC4533 Data S1 (XLSX). Means of the zones of inhibitions of the bacterial strain CC4533 (Sphingobium yanoikuyae strain PR86 variant) and Chlamydomonas and corresponding standard deviations.\n\nCC4533 Data S2 (XLSX). Statistical analyses of the zones of inhibitions of the bacterial strain CC4533 (Sphingobium yanoikuyae strain PR86 variant) and Chlamydomonas, induced by four antibiotics.\n\nFigshare: Images of antibiotic plates of the bacterial strain CC4533 (Sphingobium yanoikuyae PR86 strain variant partial 16S rRNA sequence; GenBank Accession # MN633285.1) and green micro-alga Chlamydomonas from the antibiotic susceptibility disc diffusion tests. https://doi.org/10.6084/m9.figshare.12465953.v145. This project contains 16 images of antibiotic plates used for the antibiotic susceptibility tests using the disc diffusion method for Chlamydomonas and the bacterial strain, CC4533 (Sphingobium yanoikuyae PR86 strain variant).\n\nFigshare: Growth of the bacterial strain CC4533 (Sphingobium yanoikuyae PR86 strain variant partial 16S rRNA sequence; GenBank Accession # MN633285.1) and Staphylococcus aureus on Tryptic Soy agar medium containing 5% sheep blood. https://doi.org/10.6084/m9.figshare.12478544.v146. This project contains six images that show the growth of the bacterial strain CC4533 (Sphingobium yanoikuyae PR86 strain variant) and Staphylococcus aureus on Tryptic Soy agar medium plates containing 5% sheep blood over a period of 3 days at 30°C.\n\nFigshare: Tests using Tris-Phosphate medium (TP) to see if hydrocarbons, aromatic compounds and polyhydroxyalkanoates can be used by the bacterium CC4533 (Sphingobium yanoikuyae PR86 strain variant, partial 16S rRNA sequence; GenBank Accession # MN633285.1) as the sole carbon source. https://doi.org/10.6084/m9.figshare.12465989.v148. This project contains 21 images of TP (Tris-Phosphate) medium plates containing different alternative carbon sources. Bacterium CC4533 (Sphingobium yanoikuyae PR86 strain variant) was streaked on these chemical plates to test if CC4533 can utilize these chemicals as the sole carbon source for energy and growth.\n\nFigshare: 16S rRNA partial gene sequences of the bacterial strain CC4533 (Sphingobium yanoikuyae PR86 strain variant partial 16S rRNA sequence; GenBank Accession # MN633285.1). https://doi.org/10.6084/m9.figshare.12466043.v152.\n\nThis project contains the following underlying data:\n\nAbi extension files obtained from partial sequencing of the 16S rRNA gene of CC4533 strain (Sphingobium yanoikuyae PR86 strain variant).\n\nCorresponding text files of DNA sequences\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nWe would like to thank: Dr. Krishna K. Niyogi (Professor, Department of Plant & Microbial Biology, UC Berkeley) for giving us the Chlamydomonas 4A+ strain; Dr. Farooq A. Khan (Professor, Chemistry Department, University of West Georgia,) for providing us with naphthalene and, Mr. Joseph H. Douglas (Lab Coordinator, Biology Department, University of West Georgia) for providing us with fresh and used 10W30 car motor oil.\n\n\nReferences\n\nYabuuchi E, Yano I, Oyaizu H, et al.: Proposals of Sphingomonas paucimobilis gen. nov. and comb. nov., Sphingomonas parapaucimobilis sp. nov., Sphingomonas yanoikuyae sp. nov., Sphingomonas adhaesiva sp. nov., Sphingomonas capsulata comb. nov., and Two Genospecies of the Genus Sphingomonas. Microbiol Immunol. 1990; 34(2): 99–119. PubMed Abstract \| Publisher Full Text\n\nTakeuchi M, Sawada H, Oyaizu H, et al.: Phylogenetic evidence for Sphingomonas and Rhizomonas as nonphotosynthetic members of the alpha-4 subclass of the Proteobacteria. Int J Syst Bacteriol. 1994; 44(2): 308–14. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "68971", "date": "03 Sep 2020", "name": "David Dewez", "expertise": [ "Reviewer Expertise Environmental toxicology and chemistry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis research was well performed, and the paper explains in detail the obtained results and the outcome.\nThis work provides original results and contributes significantly to the domain of environmental microbiology. The paper is a little bit long, but the quality is not affected. Overall, the structure of this article is good, and this research work is well supported by an extensive bibliography. However, I do not recommend using the term “dose”, since in toxicology domain it is use for the quantity absorbed by an organism per his weight (for example in µg/Kg). It may bring confusion for readers. In addition, the label of Figure 2-D should be corrected for 100 µg/mL instead of 50 µg/mL, and the label of Figure 17-B should be corrected for 2 mL instead of 0.5 mL.\n\nFinally, I approve the publication of this research article.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "68970", "date": "07 Sep 2020", "name": "Arun Karnwal", "expertise": [ "Reviewer Expertise Environment Microbiology", "Agricultural 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 manuscript is well written, has important data related to be used in future in bioremediation of hazardous saturated hydrocarbons using Sphingobium yanoikuyae and should be of great interest to the readers.\nIntroduction: it is written very well and comprises the basic information regarding bacterial species, method applied during research.\nMethods: methodology used for characterisation of bacterial strain is appropriate i.e. microscopic, macroscopic and molecular.\nResults and Discussion are presented properly and discussed with appropriate earlier work. Overall, it is an important study, and should be considered for indexing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-767
https://f1000research.com/articles/9-317/v1	01 May 20	{ "type": "Method Article", "title": "A miRNA screen procedure identifies garz as an essential factor in adult glia functions and validates Drosophila as a beneficial 3Rs model to study glial functions and GBF1 biology", "authors": [ "Catarina Gonçalves-Pimentel", "David Mazaud", "Benjamin Kottler", "Sandra Proelss", "Frank Hirth", "Manolis Fanto", "Catarina Gonçalves-Pimentel", "David Mazaud", "Benjamin Kottler", "Sandra Proelss", "Frank Hirth" ], "abstract": "Invertebrate glia performs most of the key functions controlled by mammalian glia in the nervous system and provides an ideal model for genetic studies of glial functions. To study the influence of adult glial cells in ageing we have performed a genetic screen in Drosophila using a collection of transgenic lines providing conditional expression of micro-RNAs (miRNAs). Here, we describe a methodological algorithm to identify and rank genes that are candidate to be targeted by miRNAs that shorten lifespan when expressed in adult glia. We have used four different databases for miRNA target prediction in Drosophila but find little agreement between them, overall. However, top candidate gene analysis shows potential to identify essential genes involved in adult glial functions. One example from our top candidates’ analysis is gartenzwerg (garz). We establish that garz is necessary in many glial cell types, that it affects motor behaviour and, at the sub-cellular level, is responsible for defects in cellular membranes, autophagy and mitochondria quality control. We also verify the remarkable conservation of functions between garz and its mammalian orthologue, GBF1, validating the use of Drosophila as an alternative 3Rs-beneficial model to knock-out mice for studying the biology of GBF1, potentially involved in human neurodegenerative diseases.", "keywords": [ "miRNA", "glia", "Drosophila", "screens", "GBF1" ], "content": "\n\nThis screen has provided a thorough analysis of glial functions in ageing\n\nPotential to shortcut gene discovery through miRNAs effect. The screening of only ~200 mutant lines potentially targets >6000 genes.\n\nPotential to identify complex regulatory networks that include miRNAs and target genes.\n\nValidated the identification of essential genes for the adult nervous system and their functions specifically in motor control.\n\nAn open-access searchable database for future discoveries upon improved precision of miRNA-target predictions.\n\nThis screening method provides an alternative approach for studying genes important in glial biology, without the need for animal experiments.\n\nExample validation that Drosophila can be used to study the biology of GBF1, instead of in vivo vertebrate animal models, such as zebrafish or mouse.\n\nThe searchable database can be easily updated upon emergence of updated miRNA target predictions.\n\nRNAi lines are publicly available from the Vienna Drosophila Resource Centre stock collection.\n\nGenetic studies in Drosophila are quicker and more sophisticated compared to vertebrate studies. They also maintain high conservation of functions.\n\nUncovered the function of garz in glial cells for membrane trafficking, autophagy and mitochondria quality control.\n\nStudy of genes, such as GBF1, involved in ageing and neurodegeneration\n\nIdentification of novel miRNA targets in glia\n\nStudy of novel miRNA targets in glia\n\nStudy of glial functions in controlling lifespan and healthspan\n\n\nIntroduction\n\nDespite the fact that glial cells were initially identified simply as the connective tissue of the brain1, work developed in the past decades has shed a light on a much more intricate role for these cells in developing and maintaining nervous system homeostasis (reviewed in 2). From neuronal nutrient supply3, to neurotransmitter recycling4–6, to being the first line of immune response in the brain7, glial cells have been shown to actively contribute to the correct functioning of the brain.\n\nMore recently, several studies have been taking advantage of Drosophila’s powerful genetic manipulation to better understand the role of glia in the development and maintenance of the nervous system (see 8 for review).\n\nThe use of invertebrate models is also a powerful 3Rs solution to reduce and replace animal experiments. It expressly applies to complex matters in which cross-talk between different cell types (e.g. glia and neurons) is a focal point of the investigation, given that these complex environments are more difficult to model in vitro and in silico. Popular animal models for studying glial functions are zebrafish, which provide a useful platform for tissue and cell biology, with some capability for genetic manipulation9 and genetically modified mice10. Despite having a different developmental origin, glial cells have converged in Drosophila and mammals towards the same key functions of neurotransmission regulation, insulation and immune surveillance/phagocytosis8, making the fruit-fly an organism of choice for studying the function of glial cells.\n\nWe have tackled the functions of glial cells in ageing. We have previously screened a large collection of miRNAs regarding their effects on Drosophila’s lifespan upon ectopic expression in glial cells in adult flies and have validated this screen through the analysis of repo, an already-established key glia gene11. The experimental advantage of performing a miRNA-based screen followed by in silico identification and ranking of predicted miRNAs target transcripts11,12 has, however, its bottleneck in the validation of the action of the genes of interest. In principle, the specific knockdown of predicted target genes should mimic, to some extent, the phenotype obtained upon corresponding miRNA overexpression.\n\nIn fact, using databases of predicted miRNA-target genes previously allowed us to identify repo as an important player for maintaining glial function and, consequently, homeostasis in the adult brain11. We have shown that while the miR-1-repo axis is physiologically relevant only in the embryo during the glia versus haemocyte cell fate choice13, the miRNA-target relationship can be exploited as a discovery tool to identify the functions of a target gene in a different context, namely adult glial functions11.\n\nWhile the focus on repo was based on its already-established role in glia cell function, here we attempt a global and unbiased systematic in silico approach. In order to systematically identify potential target genes that could account for the lifespan phenotype, focusing on the miRNAs that shortened lifespan, we set out to devise a quantitative algorithm. The aim of this algorithm is to identify and rank the predicted target genes so that those ranking on top would be the most relevant for adult glia in lifespan and ageing.\n\nThis is followed by experimental validation of the function of these targets in adult glia in the same paradigm used in the miRNAs screen.\n\nWe conclude that this approach is valid but has issues of efficiency given the large number of predicted targets that do not recapitulate the expected phenotype. We also establish that there is no significant synergy generated by focusing on the common predictions between all available miRNAs target databases. Nevertheless, the main outcome of our work is a list of candidate genes whose function is essential in glial cells during ageing. These genes can be studied in the future in Drosophila, with the tools identified here, rather than in genetically modified mouse models or in zebrafish, providing an incentive towards animal replacement and reduction and advancing the 3Rs. Mouse and zebrafish neuroscientists and geneticists could take advantage of this information to test preliminary approaches and exploratory experiments in Drosophila, prior to validation in their system reducing the number of animals used. Alternatively, they may entirely replace vertebrate animals with Drosophila to study highly conserved genes and glial functions.\n\nThe success of this in silico approach is exemplified by our analysis of one of the top predicted targets: gartenzwerg (garz), the fly orthologue of GBF1 (golgi brefeldin A resistant guanine nucleotide exchange factor 1), a small GTPase guanine exchange factor. Here, we show that garz is an essential factor in glia homeostasis maintenance.\n\nSmall GTPases regulate a wide range of cellular events such as proliferation, morphology, nuclear transport and vesicle formation14. The conversion from GDP-bound (inactive) to GTP-bound (active) forms of these enzymes relies on the activity of GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). While GAPs are responsible for their inactivation through GTP hydrolysis, GEFs are responsible for their activation promoting the exchange of GDP by GTP15.\n\nGEFs belonging to the Sec7 domain protein family are responsible for the activation of Arf (ADP-ribosylation factor) GTPases which are associated with the recruitment of coat proteins (COP) to vesicle budding sites16–18. GBF1 is part of this family19 and is highly conserved in all eukaryotes, conferring significant translatability of the findings obtained using different model organisms.\n\nStrongly localized in the cis-Golgi compartment, GBF1 has been shown to regulate vesicle trafficking between the endoplasmic reticulum (ER) and the Golgi apparatus20–24. Mutated versions or knock-down of garz expression brings about epithelial morphogenesis defects during development conditioning embryonic trachea and larval salivary gland formation20,21. Additionally, in accordance with a role in membrane delivery and vesicular trafficking, silencing of garz in these glands impairs membrane delivery of adhesion molecules25. Independently from its role in secretion, GBF1/garz has also been implicated in pinocytosis26; intestinal stem cell survival27; cell cycle28,29; unfolded protein response events29; mitochondria morphology and function30; and autophagy31,32.\n\nHere we show that garz knock-down resulted not only in lifespan reduction but also in motor deficits of adult flies and in subcellular phenotypes indicative of dysfunctions in trafficking, autophagy and mitochondria. Additionally, miRNAs overexpression and garz knockdown phenotypes were reverted by expression of its mammalian orthologue GBF1, stressing the conservation of functions and the appropriateness of using Drosophila in place of vertebrate models to study the biology of GBF1.\n\n\nMethods\n\nThe following databases were used for the prediction of miRNA targets:\n\nMicroCosm (https://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/)\n\nmicroRNA.org (http://www.microrna.org/microrna/home.do)\n\nTargetScan (http://www.targetscan.org/fly_72/)\n\nPicTar (https://pictar.mdc-berlin.de/)\n\nEach of the databases provides for every miRNA a numerical prediction of the likelihood of targeting a given gene (Score). For MicroCosm and PicTar this was used without additional steps. In the case of miRNA.org this score is a negative value and we have squared it to obtain a positive number. In the case of TargetScan a numerical score was calculated on the basis of the information provided by the database as follows: conserved 8mer = 10 points, conserved 7mer-m8 = 6 points, conserved 7mer-1A = 4 points, poorly conserved 8mer = 8 points, poorly conserved 7mer-m8 = 4 points and poorly conserved 7mer-1A = 2 points. A detailed explanation of the 8mer and 7mer species can be found on the TargetScan website and in the original publication33.\n\nThe algorithm for ranking targets within each database consists of two steps.\n\nStep 1 - column (Score)Av(χ2) or (Score2)Av(χ2):\n\nFor each miRNA, every target score (or its square value) was multiplied by the Average Chi square (χ2) obtained in the miRNAs screen (from Table 1). Information regarding different mRNAs for the same gene, where available, was grouped under the same gene name\n\nStep 2 - column Σ(Score)Av(χ2)] or Σ(Score2)Av(χ2)]:\n\nFor each target gene, as defined by its CG number/accession ID, all values resulting from all miRNAs predicted to target the same gene were summed in a final ranking value. Information regarding different mRNAs from the same gene, where available, was grouped under the same gene name.\n\nThe algorithm for comparing the ranking between different databases and providing a final common ranking consists of two steps:\n\nStep1 - column Normalised Σ[(Score)Av(χ2)] or Normalised Σ[(Score2)Av(χ2)]\n\nFor each database the Σ(Score)Av(χ2)] was normalised to 100 and then weighted for the fraction of miRNAs present in the database, out of the total tested in our miRNAs screen. For TargetScan the groups of miRNAs families were counted as one unit in each case.\n\nStep 2 – column Σ{Normalised Σ[(Score(2))Av(χ2)]} For each target gene, all values from all databases were summed into a final ranking number.\n\nTo determine the strength of miRNAs in our lifespan assay we have used the averaged χ2 values from each transgenic line used in our previously published analysis11. When only one line was tested for a given miRNA, the value was divided in half, i.e. assuming a neutral value of 0 for a second putative untested line. For the TargetScan database, some miRNAs are grouped in families requiring an amendment to our approach. In this case, we have averaged all miRNAs in the given families. Additionally, some of the lines tested for these grouped miRNAs had, in the original screen the opposite effect of what is here considered, i.e. extending lifespan with respect to the control used. To account for this opposite effect the χ2 values for these miRNAs have been given negative values and have been effectively subtracted, when calculating the Av(χ2) parameter.\n\nFlies were kept on standard cornmeal agar food (0.8% w/v agar, 2% w/v cornmeal, 8% w/v glucose, 5% w/v Brewer’s yeast, 1.5% v/v ethanol, 0.22% v/v methyl- 4-hydroxybenzoate, 0.38% v/v propionic acid) at 18°C or room temperature. Unless stated otherwise, w1118 flies were used as control. The following lines were acquired from the Bloomington collection: w1118 (RRID:BDSC_3605), repo-Gal4 (RRID:BDSC_7415), NP2222-Gal4 (RRID:DGGR_112830), moody-Gal4, elav-Gal4 (RRID:BDSC_8765), tub-Gal80ts (RRID:BDSC_7019). alrm-Gal4 (RRID:BDSC_67031) was kindly provided by M. Freeman (University of Massachusetts) ; UAS-miR-1, UAS-miR-79 and UAS-miR-315 were generated by E. Lai (Sloan Kettering Institute) for the miR library34; UAS-garz-RNAi (42140/GD and 42141/GD) as well as all RNAi lines used are from Vienna Drosophila Resource Center (VDRC); gliotactin-Gal4 was provided by R. Sousa-Nunes; UAS-mito-GFP was provided by J. Bateman; UAS-garz; UAS-garz Sec7-; UAS-GBF1 and UAS-ΔGBF1Sec7- were kindly provided by S. Luschnig.\n\nLifespan analysis was performed as previously described35. Briefly, crosses were maintained at 18°C throughout the whole development of the progeny. Within the first 5 days post-eclosion, adult flies were collected, and female and male flies pooled together. An equal number of flies was distributed in three vials, a total of 60 flies was used. This group size has a power of 0.8 in one tailed survival test at 50% survival for the control group and 29% for an experimental group at 0.05 significance. Lifespan assessment was performed in a controlled environment of 29°C and 60% humidity, three times a week. Upon short CO2 anaesthesia (5 s), the number of dead vs alive flies was counted, and the alive flies transferred into a fresh vial.\n\nSingle fly tracking was carried out as previously described11. In each experiment, up to 20 flies per genotype were placed into individual glass tubes. This group size has a power of 0.9 and significance 0.05 for three groups with an effect size of 0.48, as measured for the mean bout length. All the genotypes were positioned on the same platform, having two shaft-less motors placed underneath each subplatform containing each, one genotype. The protocol used consisted of 6 stimuli events equally split during a period of 2 h and 15 min, the first one starting after 30 min of recording and the last one 30 min before the end of the protocol. Each stimuli event was composed of 5 vibrations of 200 ms spaced by 500 ms. The x/y position of each single fly was tracked and analysed using DART software 1.0 (freely distributed upon request to info@bfklab.com) in order to evaluate the relative speed and activity before, during and after the stimuli event. The speed analysis was used for the “Stimuli Response Trace” and the general activity used to deduce “Active Speed”, “Mean Bout Length” and “Inter-Bout Interval”, using a custom-made modification of the DART software36. Raw data were analysed with GraphPad Prism for statistical significance and DART-derived graphs were edited with Adobe Illustrator CC2017 (RRID:SCR_010279).\n\nFlies (N=5–10) were briefly (5 s) anesthetized with CO2 and kept on ice, entire fly brains were dissected under a stereoscope and immediately fixed in 4% paraformaldehyde (PFA, from EMS) in Phosphate Buffer Saline (PBS) for 30 min. After washing with PBS, the brains were incubated for blocking in PBS with 0.3% triton-X (BDH 306324N) (PBT) and 10% foetal bovine serum (Sigma F4135) for 1 hr. Primary antibody incubation was done overnight at 4°C and followed by three washes (20 min each) in PBT. Secondary antibody incubation for 1hr at room temperature was followed by three washes. All steps were in 50-µl volume in a 96-well plate on a gentle rocker. Brains were then mounted on a slide in Vectashield with DAPI (Vector Labs). The following primary antibodies, diluted in blocking solution (see above): anti-Repo (1/100, mouse DSHB 8D12, RRID:AB_528448); anti-GFP (1/1000, rabbit, Life technologies, A11122) anti-GFP(1/100, mouse, Roche, RRID:AB_390913), anti-GFP (1/500, chicken, kindly provided by M. Meyer); anti-Ref(2)P (1/2000, rabbit, a gift of Tor Erik Rusten). Secondary antibodies were all from Life technologies (conjugated with Alexa-488, Alexa-555 or Alexa-666) and diluted 1/200 in blocking solution (see above).\n\nZ-stacks at intervals of 0.3 µm or 5 µm were taken at 1024×1024 pixel/inch resolution. For control vs garzIR comparisons, microscope settings were established using control flies to have a GFP signal below saturation and kept unchanged throughout all acquisitions. All images were acquired with a Leica TCS SP5 confocal microscope and mitochondria sphericity, volume and surface area in Figure 3B,C were measured using the 3D Object Counter 2.0.1 plugin37 in the ImageJ Fiji 1.52n software (RRID:SCR_002285).\n\nAll statistical analysis was performed with Graph- Pad Prism 7 software (RRID:SCR_002798). For all lifespans, the statistical analysis was performed using the log–rank test of the Kaplan and Meier method. For behavioural experiments (DART), the statistical analysis was done by one-way ANOVA using Dunnett’s multiple comparisons post hoc test. Significance is shown by asterisks in all figures as follows: P<0.05, P<0.01, P<0.001, and *P<0.0001.\n\nIn each experiment the desired number of flies were selected haphazardly from a much larger cohort of flies with the same genotype and sex. Blinding was performed in lifespan and behaviour by masking the genotypes with a numerical or alphabetical serial labelling.\n\n\nResults\n\nFirstly, such algorithm should prioritise the information for the miRNAs that had the strongest effect on the fly lifespan in our miRNA screen. To achieve this, we have quantified the average strength of each miRNA using the Chi square (χ2) of each Kaplan Mayer analysis (Table 1).\n\nTo identify potential target genes, we used four different databases available online: EBI MicroCosm, PicTar, microRNA.org and TargetScan. Each database weights the likelihood of every miRNA to target a given gene with a numerical score. Where this is different, for TargetScan, we calculated a numerical score on the basis of the sequence information provided by the database (see Methods).\n\nTherefore, to rank target genes within each database taking into account both the likelihood of being targeted by a given miRNA and the strength of the effect of this miRNA in adult glia, we first multiplied the average strength of each miRNA from our screen (values in Table 1) by the strength of the target prediction (Score) given by the database, obtaining the parameter (Score)Av(χ2). This was done for all miRNAs tested in our screen that were present in each database.\n\nBecause a given gene can be targeted by more than one miRNA, to rank its overall importance in adult glia, we have summed all the values obtained for a given gene that were calculated for different miRNAs, obtaining the parameter Σ[(Score)Av(χ2)]. In the case of TargetScan, some miRNAs are grouped in families and we have considered them as a single unit value. This underweights these miRNAs in comparison to others and the genes targeted by them (for instance a gene targeted by miR-9a, miR-9b and miR-9c would obtain a Σ[(Score)Av(χ2)] that is the sum of three (Score)Av(χ2) in the other databases, but for TargetScan it would only reflect one (Score)Av(χ2). Our reasoning was that grouped miRNAs in TargetScan was not taking into account valuable information and this should be reflected in a penalisation in the ranking.\n\nIn conclusion we have ranked target genes according to Σ[(Score)Av(χ2)] for EBI MicroCosm (Extended data Table 1)38, PicTar (Extended data Table 2)38, microRNA.org (Extended data Table 3)38 and TargetScan (Extended data Table 4)38. Surprisingly, this revealed that there was very little agreement among the four databases. The top-ranking genes obtained using the same algorithm were very different and only 5.6% (i.e. 520 genes) of target predictions were common to all four databases (Figure 1A).\n\n(A) Venn diagram referring to the data in Table 6 and illustrating the overlap between the four different databases used to predict gene targets of the miRNAs whose expression in the adult glia resulted in a significant reduction in fly lifespan. Only 520 target genes are in common among all four databases, garz falls in this group. A remarkably large number of genes as targets were uniquely predicted by the MicroCosm database. (B) Two RNAi lines against garz bring about a very significant reduction in fly lifespan in comparison to controls, when expressed in all adult glia. N=60 for each genotype, Error bars SEM, pairwise comparisons: Log-rank (Mantel-Cox) test. The full dataset can be accessed at DOI 10.17605/OSF.IO/8E3NS as part of Table 7. (C) Knock-down of garz in sub-populations of glial cells, astrocyte-like (alrm-Gal4), Cortex glia (NP2222-Gal4), sub-perineural glia (moody-Gal4), perineural and PNS glia (gliotactin-gal4) or in neurons (elav-Gal4) brings about a significant reduction in lifespan in comparison to controls. N=60 for each genotype, Error bars SEM, pairwise comparisons: Log-rank (Mantel-Cox) test. The full dataset can be accessed at DOI 10.17605/OSF.IO/HQCDG. (D) Lifespan reduction due to RNAi against garz in adult glia is rescued by an exogenous UAS-garz transgene and by a transgene expressing the human orthologue GBF1 under UAS control. Note that overexpression of garz in an otherwise wt background is highly detrimental to fly lifespan, whereas overexpression of GBF1 in a wt background has no adverse effects. Mutations leading to a non-functional Sec7 domain eliminate or drastically reduce the ability of garz or GBF1 transgenes to rescue fly lifespan. N=60 for each genotype, Error bars SEM, pairwise comparisons: Log-rank (Mantel-Cox) test. The full dataset can be accessed at DOI 10.17605/OSF.IO/5RGEF. (F) Co-expression of human GBF1 significantly extends the short lifespan caused by overexpression of miR-1, miR-79 and miR-315 in adult glia. N=60 for each genotype, Error bars SEM, pairwise comparisons: Log-rank (Mantel-Cox) test. The full dataset can be accessed at DOI 10.17605/OSF.IO/B37DF.\n\nTo rank these common targets for their predicted overall relevance in adult glia in ageing, we have devised additional steps. First, to make the numerical rankings from each database comparable, we have calculated the Normalised Σ[(Score)Av(χ2)] parameter by normalising the maximum value to 100. Additionally, we have weighted this number for the fraction of miRNAs present in each database, out of the total tested in our miRNAs screen. Out of 44 miRNAs screened, 31 were present in EBI Microscosm, 28 in PicTar, 43 in microRNA.org and 40 in TargetScan. The rationale for this weighting was to prioritise the databases carrying more information that was relevant to our screen. Then, for each target gene, we have combined all these scores from the four databases generating the final parameter Σ{Normalised Σ[(Score(2))Av(χ2)]} for all targets, including the 520 that were commonly predicted by all databases (Table 2).\n\nThe ranking scores from all four databases were pooled to obtain a global rank of all targets predicted by our analysis and a list of targets that are predicted by all four databases. Because the different databases contained information about some, but not all, miRNAs analysed in our screen we have weighted the completeness of each database by normalising the Σ[(Score)Av(χ2)] by the fraction of miRNAs listed in the database, out of the ones tested in our screen. In addition, to make the ranking from each database equally valued in this analysis, we have normalised each score to 100 as a maximum possible value for each database – column Normalised Σ[(Score)Av(χ2)]. Thereafter, all values for each target have been added – column Σ{Normalised Σ[(Score)Av(χ2)]} – for each target and for a specific list of 520 targets that have been predicted by all four databases, albeit with different scores. Only the top 30 rows are shown here. The full table can be accessed at https://doi.org/10.17605/OSF.IO/QWUAY.\n\nStrength of the effect on fly lifespan of RNAi lines against some of the gene targets predicted by all four databases. For most genes two different RNAi lines have been tested. In red are the lines that, in agreement with the miRNA prediction, shorten the lifespan, in comparison to controls (w1118) when specifically expressed in the adult flies with repo-Gal4 and tub-Gal80ts. In green are the lines that have the opposite effect and prolong lifespan. In black are the lines that had no effect. Highlighted in yellow are the genes for which all lines tested had the same effect and shortened lifespan. Highlighted in pink is the gene for which all lines tested had the same effect and prolonged the lifespan. To combine the target RNAi strength with the strength of the prediction we have averaged the χ² for each miRNA according to the same rules followed in Table 1 and multiplied it for the final score from Table 2 – column Σ{Normalised Σ[(Score)Av(χ²)] {Av(χ²)IR}. The top rank was achieved by garz, which was also one of the 6 genes with all RNAi lines tested having the same effect. We have also repeated the same procedure separately for the four different databases using the final normalised score (Normalised Σ[(Score)Av(χ²)]) obtained from each database in Tables 2, 3, 4 and 5, and also reported in Table 2– columns Normalised Σ[(Score)Av(χ²)] {Av(χ²)IR} for each database. The predicting power for the combination of all databases and for each database sums all scores in this column to quantify the global predicting value of each database in comparison to their combination. This is further normalised by the number of predicted targets for the whole screen, to measure the efficiency of each database when predicting target genes. The full dataset can be accessed at DOI 10.17605/OSF.IO/8E3NS.\n\nStrength of the effect on fly lifespan of RNAi lines against some of the gene targets predicted by some, but not all, databases. In red are the lines that, in agreement with the miRNA prediction, shorten the lifespan, in comparison to controls (w1118) when specifically expressed in the adult flies with repo-Gal4 and tub-Gal80ts. In green are the lines that have the opposite effect and prolong lifespan. In black are the lines that had no effect. Highlighted in yellow is the gene for which all lines tested had the same effect and shortened lifespan. To combine the target RNAi strength with the strength of the prediction we have averaged the χ² for each miRNA according to the same rules followed in Table 1 and multiplied it for the final normalised score (Normalised Σ[(Score)Av(χ²)]) obtained from each database in Extended data Tables 1, 2, 3and 4 [49], and also reported in Table 2 – columns Normalised Σ[(Score)Av(χ²)] {Av(χ²)IR} for each database. The predicting power for each database sums all scores in this column to quantify the global predicting value of each database. This is further normalised by the number of predicted targets for the whole screen, to measure the efficiency of each database when predicting target genes, as in Table 3. The full dataset can be accessed at DOI 10.17605/OSF.IO/QTASN.\n\nTo test these predictions, we decided to screen for the lifespan effect, a number of RNAi lines from Vienna Drosophila Resource Center (VDRC) that were already present in our stock collection. These corresponded to a random selection of approximately 10% (51 out of 520) of commonly predicted target genes. Adopting a similar strategy used for the miRNA screen, we have used the repo-Gal4, tub-Gal80ts inducible system to trigger the RNAi expression in all glial cells in adult flies. As negative control, we used the offspring of crossing repo-Gal4, tub-Gal80ts to w1118 throughout the screen. The expectation was that RNAi against these target genes in adult glia, would phenocopy the effect of the miRNAs that are predicted to target them, therefore shortening lifespan.\n\nThe gold standard commonly used by the Drosophila community to gain confidence about the effects of RNAi knock-down is to obtain a similar effect when testing two RNAi lines against the same gene (2-RNAi lines criterion). Remarkably, only in six cases at least two different RNAi lines tested for the same gene delivered the shorter lifespan phenotype that was predicted (Table 3). In another case both RNAi lines tested had the same effect, but it was the opposite of the predicted one, extending lifespan with respect to the control flies.\n\nIn other cases (11/51) there was an overall confirmation of the prediction, but the two RNAi lines tested for one given target did not share the same effect or we were able to test only one line. The largest group (19/51) was made by cases in which there was no effect and surprisingly in a remarkable number of cases (14/51) there was an overall effect opposite to that predicted, albeit either the two RNAi lines tested for one given target did not share the same effect or we were able to test only one line.\n\nIn addition to the 2-RNAi lines criterion we have devised a quantitative index for ranking these targets by combining their effect in the RNAi screen (averaging the Chi square for the RNAi lines targeting each gene, Av(χ2)IR) with the strength of the prediction in all combined databases (Σ{Normalised Σ[(Score)Av(χ2)]}).\n\nThis parameter (Σ{Normalised Σ[(Score)Av(χ2)]}{Av(χ2)IR}) highlighted garz, one of the six targets satisfying the 2-RNAi lines criterion, as the top target (Table 3). However, there was incomplete agreement with respect to the rest of the ranking between the two criteria, i.e. our scoring system and the rule of 2-RNAi lines, with only four of the ten top scores coming from target genes satisfying the 2-RNAi lines criterion.\n\nWe also tested 14 additional targets that were differentially predicted by the different databases. We were able to further identify five targets that confirmed the predicted phenotype, one satisfying also the 2-RNAi lines criterion, while two had the opposite overall effect (Table 4).\n\nA comparison between these two groups, the common to all databases and the differentially predicted, highlights that the fraction of validated prediction is similar, but the chance of finding false positives (i.e. targets that had the opposite effect to that predicted) is paradoxically higher in the commonly predicted group (15/51 in the common and 2/14 in the differential).\n\nConsidering the lack of tangible benefits of focusing on the commonalities between the different databases, we have then exploited our validation analysis to quantify the prediction capability of each of the four databases to identify the most valid for our screen. For all targets tested, both from the common group (Table 3) and from the differential group (Table 4), we have calculated the database-specific Normalised Σ[(Score)Av(χ2)] {Av(χ2)IR} parameter by combining the quantification of the lifespan effect of the RNAi lines (average Chi square in the RNAi screen) with the normalised predicted score from each database. Then, to rank databases we have summed all these results (with a negative value for false positives) to determine the predicting power score. TargetScan had the highest predicting power for the list of common targets, while MicroCosm had the highest capacity for target identification among the differential targets. PicTar had the lowest predicting power in all cases. However, MicroCosm also predicted the largest number of genes as targets of our miRNA screen, with over 44% of them not shared by the other databases. We reasoned that this lack of efficiency in EBI Microcosm had to be considered and when normalising for the total number of predicted targets from each database, as a measure of the predicting power efficiency, TargetScan showed a greater efficiency in both cases, followed by miRNA.org.\n\nAs mentioned, we ranked the target genes from the RNAi confirmed predictions and decided to further investigate the top ranked target, garz, the fly orthologue for GBF119,20,39).\n\nPan-glial knockdown of garz with repo-Gal4 specifically during adulthood strongly reduced lifespan. This was true for both RNAi lines tested when compared to w1118 median lifespan control (Figure 1B). Different glial cell types present in the adult fly brain have specific morphology and function40. In order to test if a specific glial sub-population could account for the observed phenotype, we targeted the knockdown of garz using established Gal4 driver lines: astrocyte-like (alrm-Gal4), cortex (NP2222-Gal4), subperineural (moody-Gal4) and peripheral (gliotactin-Gal4) glia. In all sup-populations tested, the downregulation of garz caused a reduction in lifespan, albeit not as strong as the pan-glial knockdown (Figure 1C). This suggests that a combination of multiple functions is affected by garz.\n\nWe also analysed the effects of pan-neuronal (elav-Gal4) knockdown of garz. This also led to a significant shortening of lifespan although the effect was milder than the one obtained with pan-glial garz knockdown (Figure 1B vs 1C), either because of differences in Gal4 line strength or because of a higher impact of garz function in glial cells for maintenance of the brain homeostasis.\n\nWe then focused on rescuing the glia-related shorter lifespan phenotype using exogenous transgenes for garz and human GBF1. Although the overexpression of garz alone in adult glia had a very toxic effect, when combined with the garz-RNAi overexpression, promoted a modest but significant rescue of the lifespan (Figure 1D). This suggests that garz levels need to be tightly controlled in the fly. On the other hand, overexpression of the human GBF1 was entirely neutral for fly lifespan when expressed on its own and fully rescued the lifespan phenotype when co-expressed with garz RNAi. This indicates a remarkable conservation in functions between garz and GBF1.\n\nFor both garz and GBF1, the presence of a functional Sec7 domain, which is responsible for the catalytic activity of GEF proteins domain24, was important to exercise their rescue activity (Figure 1D). In the case of UAS-garz, a mutation of the Sec7 domain entirely eliminated the rescue of garz knock down, actually aggravating toxicity. This also indicates that the toxicity of garz overexpression is not dependent on the catalytic GEF function of garz, possibly suggesting a dominant negative effect by sequestration of binding partners in catalytically inactive complexes. Additionally, in the case of UAS-GBF1, the rescue effect was significantly reduced, albeit not entirely eliminated, by an inactive Sec7 domain (Figure 1D).\n\nHuman GBF1 showed a remarkable capability to fully rescue lifespan shortening upon garz knockdown in glia. We then asked whether it would also be able to rescue the lifespan shortening induced by miRNAs predicted to target garz. From our database analysis, miR-1, miR-79 and miR-315, all causing a strong reduction of lifespan11, were among the miRNAs predicted to target garz and may be rescued by GBF1. Indeed, UAS-GBF1 was able to significantly rescue the phenotypes caused by the overexpression of these miRNAs in glia (Figure 1E). GBF1 co-overexpression was able to rescue the lifespan for miR-79 and miR-315 to what would be commonly observed in wild-type flies. These results confirm our initial predictions and establish garz as the main mediator of the effect on lifespan caused by overexpression of miR-79 and mir-315 in adult glia. The partial rescue of the miR-1 phenotype indicates that garz is only partially responsible for the effect of miR-1 in adult glia and is in accordance with the previously reported role of repo in miR-1-mediated lifespan shortening11.\n\nWe have previously described an automated unbiased and high-throughput method to analyse fly motor activity11). When using this paradigm, we unravelled an impact of glial garz knockdown on the amplitude of the response to a train of stimuli and GBF1 co-overexpression rescued this response (Figure 2A). When looking at spontaneous activity parameters, i.e. non-stimulus driven, in the same experiment, flies expressing garz-RNAi showed a reduced average speed and an increased interval between bouts of movement without reflecting in the overall bout movement duration. Both average speed and inter-bout interval were fully rescued by the co-overexpression of GBF1 (Figure 2B–D). This analysis indicates that garz knock down affects not only lifespan but also the healthspan and motor activity both exogenously stimulated and internally generated, making flies slower and also pausing more.\n\nAll data in this figure represent a grouping of two independent experiments with a total number of flies analysed (N) of 35–40. Error bars represent SEM in all graphs. Untreated track data can be accessed at DOI 10.17605/OSF.IO/UNJX7. (A) Stimulus response curve for control flies (black), garz RNAi (red) and co-expression of GBF1 and garz RNAi (green). The graph is an average of 6 tracks for each of the stimuli received at 15 min intervals (See Methods). All genotypes also include repo-Gal4 and ubi-Gal80ts to express the transgenes in all adult glia. In control flies the presence of tub-Gal80 blocks any expression of UAS-transgenes. The graph to the right reports the mean amplitude of the response to a train of stimuli, which is significantly reduced by RNAi against garz, and this reduction is reverted to normal level by co-expression of human GBF1. One-way ANOVA, Dunnett’s multiple comparisons post hoc test. (B) Average speed analysis of the same flies as in A. RNAi against garz significantly slows down fly motility and this is rescued by human GBF1. One-way ANOVA, Dunnett’s multiple comparisons post hoc test. (C) Mean bout length analysis of the same flies as in A. No significant difference is detected in this parameter. One-way ANOVA, Dunnett’s multiple comparisons post hoc test. (D) Mean interbout interval analysis of the same flies as in A. RNAi against garz significantly increases the time spent in inactivity by flies and this is rescued by human GBF1. One-way ANOVA, Dunnett’s multiple comparisons post hoc test.\n\nWe next set out to determine the effects garz knock down had inside the glial cells that would correlate with behavioural and lifespan dysfunctions.\n\nIt has been reported that garz knockdown impairs vesicle transport and membrane delivery during fly development25. Thus, we analysed membrane distribution in the presence of garz-RNAi in adult brains. Driving the expression CD8-GFP in glia showed aberrant membrane distribution upon garz knockdown when compared to a more homogeneous distribution of the GFP signal in glia from control brains (Figure3A, Videos 1 and 2). Such data suggests that overall membrane trafficking in glia may be impaired although we have not been able to detect failure in membrane delivery of the cell adhesion cadherin molecule CadN (data not shown, the full dataset can be accessed at DOI 10.17605/OSF.IO/7HRZS).\n\n(A) Representative single confocal sections of adult fly brains stained for DAPI (blue), GFP (green), Repo (magenta) and Ref (2)P (red). Pan glial knock-down of garz with repo-Gal4 and ubi-Gal80ts leads to abnormal distribution of the plasma membrane targeted CD8-GFP protein (expressed from a UAS-CD8-GFP transgene in all glial cells) leading to gaps and blebs (arrows, see also Videao1 and 2), and to accumulation of Ref(2)P puncta (arrowheads). The full dataset can be accessed at DOI 10.17605/OSF.IO/96TS3. (B) Representative single confocal section of adult fly brains stained for DAPI (blue), GFP (green) and Repo (red). The GFP signal also in back and white (lower panels) is due to the presence of a UAS-mitoGFP transgenes and detects mitochondria. (C) Quantification of mitochondria parameters based on the GFP signal in B. Pan glial knock-down of garz with repo-Gal4 and ubi-Gal80ts leads to significant increases in the volume, surface area and sphericity of mitochondria. Mann-Whitney non-parametric test. N=300 objects, randomly selected from 4 brains. Error bars represent SEM. The full dataset can be accessed at DOI 10.17605/OSF.IO/EXMTG.\n\n\n\n\n\nConflicting in vitro data has been reported for the effects of GFB131 and garz32 in what concerns autophagy regulation. Looking at the distribution of the Ref(2)p (the orthologue of mammalian p62) autophagy receptor43 revealed Ref(2)p accumulation in puncta, suggesting a potential block in autophagic clearance in glial cells (Figure 3A).\n\nFinally, it has been suggested a role for GBF1 in the regulation of mitochondria morphology and function in yeast, C. elegans muscle and HeLa cells30. Using mito-GFP transgene we were able to identify mitochondrial morphology defects in adult glial cells (Figure 3B, C). Quantification of the main morphological parameters has unravelled an overall increased mitochondrial volume, surface and sphericity upon garz knockdown. These parameters may indicate a defect in mitochondria quality control and are in agreement with an impaired autophagic clearance, which has the potential to also affect mitophagy.\n\nUnderlying data contains the raw data behind these results44.\n\n\nDiscussion\n\nWe have previously screened a library of miRNAs for effects on Drosophila’s lifespan when expressed in adult glia and already established that this strategy can identify factors important for nervous system health in adult life11. We aimed here at developing a generalizable global approach that would allow to identify the key target genes that mediate the actions of miRNAs in a given context. Focusing on miRNAs that shortened the lifespan, we devised an in-silico strategy to unravel a potential list of genes relevant for glial function and consequently brain homeostasis in adult flies. The outcome of this strategy had efficiency issues and highlighted the little overlap in the predictions made on the basis of four different databases for miRNA target prediction in Drosophila.\n\nTo put to the test the outcome of these in silico predictions, we have silenced individual genes by inducing the expression of specific RNAi in adult glia. The assumption being that RNAi downregulation of the top target genes would phenocopy the effect observed when expressing the miRNAs targeting them, i.e. lifespan reduction. Overall, however, the number of genes that, upon knockdown, reduced lifespan was remarkably low, and we could observe no tangible benefit of focusing on predictions in common to all four databases, versus targets differentially predicted in the different databases. It was also evident from our analysis that, among the databases, TargetScan and miRNA.org were considerably more efficient in delivering predictions that withstood the RNAi tests.\n\nTherefore, the benefits of using miRNAs-based screens and in silico identification of targets, in place of much larger screens based on targeting single genes, have to be carefully evaluated and in silico selection of target genes should be based primarily on the TargetScan and miRNA.org databases. Nevertheless, the fraction of validated positive target genes by two criteria (7/65) and by at least one (22/65) is much larger than what usually expected in siRNA screens and suggests a 3/5-fold enrichment in positive hits. Thus, our method makes Drosophila screens a more appealing platform with reduced workload in comparison to traditional single gene targeted screens, whether by RNAi or genomic mutagenesis. This may have 3Rs benefits, facilitating the use of Drosophila as a model for preliminary studies on the genetic factors that influence a given biomedical process.\n\nOur screen has also highlighted a number of genes that are strong, and in most cases unexpected, candidates for essential functions in adult glia in ageing. This list of genes provides a useful tool for scientists studying glial functions in ageing. In particular, all identified genes that have been validated by two RNAi lines have clear mammalian orthologues. Drosophila can therefore be used to study in detail the functions of these genes in the adult glial cells, in place of genetically modified mouse models.\n\nTo validate our findings, we focused on the top target of the genes commonly predicted by all databases and also by TargetScan, i.e. garz, the fly orthologue of the human GBF1.\n\nThe analysis of garz confirmed that this gene is absolutely required in adult glia, and also in neurons, for fly survival. Using our automated behavioural set up we could also establish that garz is essential in glia for locomotor activity in response to a stimulus or endogenously generated. Analysing the effects of silencing garz in different glial sub-populations showed that the strong reduction in lifespan could not be accounted for by one specific type of glia but rather due to a combined effect of silencing garz in all glial cells simultaneously, indicating that garz is essential for any glial cell type.\n\nOur subcellular analysis suggests that the locomotor and lifespan defects correlate and possibly originate from a number of cellular defects in protein trafficking, autophagy and mitochondria quality control.\n\nIn Drosophila, mutated versions or knockdown of garz resulted in developmental epithelial morphogenesis defects20,21 and impaired membrane delivery of adhesion molecules25. We have been able to identify membrane defects in glial membrane distribution, although not all membrane proteins seemed to be affected by garz knockdown. garz and GBF1 have been identified as a positive autophagy regulator in Drosophila primary cultured muscle cells32 and mammalian cells31. An accumulation of Ref(2)P upon garz-RNAi expression in adult glia suggests an autophagic clearance deficits, in agreement with these studies.\n\nGBF1-RNAi has been shown to affect mitochondrial morphology and function30. Chemical inhibition of GBF1 in mammalian cells also showed condensed mitochondria and mislocalisation in the cell45. Although mislocalisation of mitochondria is difficult to assess due to glial cell morphology in the Drosophila brain, garz-RNAi strongly affected mitochondria morphology suggesting a more condensed state which may be a reflection of an unbalanced fission/fusion regulation and mitochondria quality control46.\n\nOur analysis further suggested that there was remarkable functional conservation between garz and human GBF1, with the latter being able to fully rescue, partially in a Sec7-domain dependent manner, the shorter lifespan and motor behaviour phenotypes caused by the silencing of garz. GBF1 was also able to rescue the lifespan shortening by three different miRNAs, miR-1, miR-79 and miR-315, validating that in our screen their effect is at least partially, and in some cases almost entirely, due to downregulation of garz.\n\nThus, these data validate both the logic and principles of miRNA screens, despite inefficiencies, and the use of Drosophila as a valid organism to study the biology of garz/GBF1.\n\nThe identification of major cellular events regulated by garz/GBF127–29,47–49 has targeted such molecules for health and disease studies18. Recently, it has been shown that siRNA knockdown of GBF1 causes intracellular APP accumulation in primary cortical neurons; overexpression of GBF1 contributes to APP trafficking and is dependent on its GEF activity50. Inhibition of GBF1 with brefeldin A was also shown to lead to a new form of cellular degeneration and death in neurodegenerative diseases, based on destruction of the nuclear lamina51.\n\nGbf1 conditional mutant mice have been generated in the Wellcome Trust Sanger Institute and are being phenotyped by the International Mouse Phenotyping Consortium (https://www.mousephenotype.org/data/genes/MGI:1861607). We demonstrate here that Drosophila would constitute an ideal organism to put forward 3Rs-compliant alternatives and, at least partially, replace this mouse line in studies aiming at understanding the role of GBF1 in health and disease.\n\n\nData availability\n\nOpen Science Framework: miRNA-garz. https://doi.org/10.17605/OSF.IO/A5ZST44.\n\nThis project contains the following underlying data:\n\nTable 2 (XLSX). (The complete Table 2.)\n\nTable 2 Data – Pimental et al., 2020 (XLSX). (Data underlying Table 2.)\n\nTable 3 Data – Pimental et al., 2020 (XLSX). (Data underlying Table 3.)\n\nTable 4 Data – Pimental et al., 2020 (XLSX). (Data underlying Table 4.)\n\nFigure 1C Data - Pimentel et al., 2020 (XLSX). (Data underlying Figure 1C.)\n\nFigure 1D Data - Pimentel et al., 2020 (XLSX). (Data underlying Figure 1D.)\n\nFigure 1E Data - Pimentel et al., 2020 (XLSX). (Data underlying Figure 1E.)\n\nFigure 2 Data - Pimentel et al., 2020 (XLSX). (Data underlying Figure 2.)\n\nFigure 3A and videos. (TIFF images and ZIP files containing data underlying Figure 3A.)\n\nFigure 3B-C. (ZIP files containing raw images underlying Figure 3B, C.)\n\nExtended data Table 1- Data - Pimentel et al., 2020 (XLSX). (Data underlying Extended data Table 1.)\n\nExtended data Table 2- Data - Pimentel et al., 2020 (XLSX). (Data underlying Extended data Table 2.)\n\nExtended data Table 3- Data - Pimentel et al., 2020 (XLSX). (Data underlying Extended data Table 3.)\n\nExtended data Table 4- Data - Pimentel et al., 2020 (XLSX). (Data underlying Extended data Table 4.)\n\nData not shown. (ZIP files containing images of membrane delivery of the cell adhesion cadherin molecule CadN.)\n\nOpen Science Framework: miRNA-garz. https://doi.org/10.17605/OSF.IO/K5HW938.\n\nThis project contains the following extended data:\n\nExtended Data Table 1. MicroCosm target prediction and ranking tables. For each miRNA, ranking of target prediction - column (Score)Av(χ2) - was made by multiplying the Average χ2 obtained in the screen (from Table 1) by the Score predicted in the MicroCosm database. In the total table, all values from a given target, resulting from all miRNAs were summed in a final ranking value in column Σ(Score)Av(χ2)]. This table and the full dataset can be accessed at DOI 10.17605/OSF.IO/R3ZX9.\n\nExtended data Table 2. PicTar target prediction and ranking tables. For each miRNA, ranking of target prediction - column (Score)Av(χ2) - was made by multiplying the Average χ2 obtained in the screen (from Table 1) by the Score predicted in the PicTar database. In the total table, all values from a given target, resulting from all miRNAs were summed in a final ranking value in column Σ(Score)Av(χ2)]. This table and the full dataset can be accessed at DOI 10.17605/OSF.IO/MDKHR.\n\nExtended data Table 3. miRNA.org target prediction and ranking tables. For each miRNA, ranking of target prediction - column (Score2)Av(χ2) - was made by multiplying the Average χ2 obtained in the screen (from Table 1) by the square value of Score predicted in the miRNA.org database. The square value was used in this case as the scoring system used by miRNA.org delivers negative values, differently from the other databases. In the total table, all values from a given target, resulting from all miRNAs were summed in a final ranking value in column Σ(Score2)Av(χ2)]. This table and the full dataset can be accessed at DOI 10.17605/OSF.IO/539J8.\n\nExtended data Table 4. TargetScan target prediction and ranking tables. The TargetScan database does not provide a scoring system for its predictions, rather a list of 8mer or 7mer sequences matched by the miRNA on the target and an information on the conservation of these sequences. We have attributed a numerical score to these sequences privileging the importance of 8mer vs 7mer and of conservation according to the scheme described in the Methods section. For each miRNA, ranking of target prediction - column (Score)Av(χ2) - was made by multiplying the Average χ2 obtained in the screen (from Table 1, some values specifically generated averaging all miRNA grouped in a single family by TargetScan) by the Score obtained according to our above-mentioned scheme. In the total table, all values from a given target, resulting from all miRNAs were summed in a final ranking value in column Σ(Score)*Av(χ2)]. This table and the full dataset can be accessed at DOI 10.17605/OSF.IO/WD6ZR.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nWe thank M. Freeman, E.C. Lai, R. Sousa-Nunes, J. Bateman, S. Luschnig, the VDRC, the DSHB and the BDSC for fly stocks and reagents.\n\n\nReferences\n\nSomjen GG: Nervenkitt: notes on the history of the concept of neuroglia. Glia. 1988; 1(1): 2–9. PubMed Abstract \| Publisher Full Text\n\nAllen NJ, Lyons DA: Glia as architects of central nervous system formation and function. Science. 2018; 362(6411): 181–185. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarcía-Cáceres C, Quarta C, Varela L, et al.: Astrocytic Insulin Signaling Couples Brain Glucose Uptake with Nutrient Availability. Cell. 2016; 166(4): 867–880. 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PubMed Abstract \| Publisher Full Text\n\nFanto M: miRNA-garz. 2020. http://www.doi.org/10.17605/OSF.IO/K5HW9\n\nPipaliya SV, Schlacht A, Klinger CM, et al.: Ancient complement and lineage-specific evolution of the Sec7 ARF GEF proteins in eukaryotes. Mol Biol Cell. 2019; 30(15): 1846–1863. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKremer MC, Jung C, Batelli S, et al.: The glia of the adult Drosophila nervous system. Glia. 2017; 65(4): 606–638. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFanto M: Video 1. CD8-GFP in control brains. 2020. f1000research.com. Media. http://www.doi.org/10.6084/m9.figshare.12162351.v1\n\nFanto M: Video 2. CD8-GFP in garz knock-down brains. 2020. f1000research.com. Media. http://www.doi.org/10.6084/m9.figshare.12162393.v1\n\nNezis IP, Simonsen A, Sagona AP, et al.: Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nViktorova EG, Gabaglio S, Meissner JM, et al.: A Redundant Mechanism of Recruitment Underlies the Remarkable Plasticity of the Requirement of Poliovirus Replication for the Cellular ArfGEF GBF1. J Virol. 2019; 93(21): e00856–19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFerlin J, Farhat R, Belouzard S, et al.: Investigation of the role of GBF1 in the replication of positive-sense single-stranded RNA viruses. J Gen Virol. 2018; 99(8): 1086–1096. PubMed Abstract \| Publisher Full Text\n\nLiu K, Liu Y, Xu Y, et al.: Regulatory role of Golgi brefeldin A resistance factor-1 in amyloid precursor protein trafficking, cleavage and Aβ formation. J Cell Biochem. 2019; 120(9): 15604–15615. PubMed Abstract \| Publisher Full Text\n\nBaron O, Boudi A, Dias C, et al.: Stall in Canonical Autophagy-Lysosome Pathways Prompts Nucleophagy-Based Nuclear Breakdown in Neurodegeneration. Curr Biol. 2017; 27(23): 3626–3642.e6. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "62978", "date": "01 Jun 2020", "name": "Ivana Bjedov", "expertise": [ "Reviewer Expertise ageing", "mTOR", "autophagy", "Drosophila" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 by Gonçalves-Pimentel et al. describes an impressive series of very elegant and demanding longevity experiments used to develop an innovative methodological algorithm to identify and rank candidate genes that are targeted by miRNAs and that shorten lifespan when downregulated in adult glial cells. The work presented offers a comprehensive comparison of different miRNA target databases and link those to the longevity analysis screen. Advantage of miRNA is targeting multiple genes at once. For instance by screening 200 miRNA lines, this examines effect of down-regulation of approximately 6000 genes. However this subsequently presents a challenge to determine which genes are targeted with a particular miRNA and which gene is accountable for a given phenotype. Therefore, here, Gonçalves-Pimentel et al. produce an algorithm in which they link their miRNA lifespan screen results to multiple miRNA databases, with the final aim to rank genes that are predicted targets by these miRNA and that affect lifespan and ageing of the glial cells. Briefly, within each data base, each target score was multiplied by values of the lifespan screen results, and then ranking value for each target gene obtain for different data bases. Values obtained from all miRNA databases for each gene were summed for final ranking. This approach combined the effect of the miRNA on lifespan with the prediction of a gene being targeted by particular miRNA, using a variety of databases to strengthen the approach. Combining different databases is particularly important given a surprising difference in their target prediction.\nThe top candidate from the screen is a gene garz, (mammalian orthologue of GBF1), for which they predict that its down-regulation in the glia shorten lifespan. Advantage of such approach is that it offers possibility for screening in an invertebrate organism to uncover genes potentially important in mammalian glia. garz is a target for three miRNA, miRNA-1, miRNA-79, and miRNA-315. The authors carefully examine its affect in different population of glial cells, replicate shorter lifespan using two different garz RNAi lines, overexpress human GBF1 to rescue short lifespan by these different miRNAs. Downregulation of garz in adult glial cells also leads to significant impairment of fly motor functions. The authors expanded their characterisation of garz-RNAi overexpressor flies further to show accumulation of Ref(2)P and likely consequent alterations/enlargement of mitochondria. Overall this is a detailed and exhaustive study. The algorithm is clearly explained and well presented, and will certainly be useful in other miRNA studies and will inspire its adaptation to other miRNA screens. Moreover the authors developed a valuable list of genes that when down-regulated in glia impact lifespan. This is a really significant resource and dataset that the authors present and should be commended for. I only have a few minor points: Were the longevity analysis done using males or females flies?\nWhy do the authors think that some of their predictions actually resulted in lifespan extension rather than shortening? Could the down-regulation of given gene have different outcomes when it occurs in concert with other miRNA gene target downregulation? Could this be commented in discussion perhaps?\nHow easily can their method be adapted for a different screen using a different output, such as for instance miRNA screen for stress resilience? In the ageing field, lifespan extension is a gold standard to detect anti-ageing genes and interventions. Could the authors comment on finding genes that extend lifespan in glia rather than shorten it?\nThe authors say” Recently, it has been shown that siRNA knockdown of GBF1 causes intracellular APP accumulation in primary cortical neurons” , could they please define APP.\n\nIn Figure 1 and 2 driver names are not very visible, could this be improved for clarity or genotypes be written in full perhaps? In Figure 3A, it is not very visible what the arrows is pointing at, at least not in my downloaded version of the article.\nOverall, this is a very valuable resource for anyone working on miRNA and glial cell. This research is an excellent example how screens in invertebrate organisms can lead to discoveries of important biological functions of mammalian orthologues.\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? Yes", "responses": [ { "c_id": "5743", "date": "23 Jul 2020", "name": "Manolis Fanto", "role": "Author Response", "response": "We thank both reviewer for their competent assessment of our manuscript. We have now provided a revised version of our paper and reply here to the questions and the issues raised by both reviewers. 1) Were the longevity analysis done using males or females flies? We apologize for not having made this clear enough but lifespan analysis was done on equal numbers of males and females. This has been stressed in the revised Methods 2) Why do the authors think that some of their predictions actually resulted in lifespan extension rather than shortening? Could the down-regulation of given gene have different outcomes when it occurs in concert with other miRNA gene target downregulation? Could this be commented in discussion perhaps? While an additive effect cannot be ruled out. All verifications were done using siRNAs against single genes, the fact that some resulted in the expected phenotype and others in the opposite phenotype than that expected is probably just due to these genes not being really targeted by the miRNA that originate the prediction, or by the fact that the effect of the miRNA is predominantly recapitulated by one of the other targets. 3) How easily can their method be adapted for a different screen using a different output, such as for instance miRNA screen for stress resilience? We believe that this method can be applied to different outputs, provided that the appropriate UAS/Gal4 combination of lines and screen methods are used. 4) In the ageing field, lifespan extension is a gold standard to detect anti-ageing genes and interventions. Could the authors comment on finding genes that extend lifespan in glia rather than shorten it? We have not focused on these genes as they were not the intended scope of our work, and we also notice that in a wt background, lifespan extension is usually much milder. A possible case of interest would be Sirt2, which mildly extends lifespan when downregulated in our screen, but we feel it would be inappropriate to speculate on this candidate gene without a deeper investigation, like the one done here for garz. 5) The authors say ”Recently, it has been shown that siRNA knockdown of GBF1 causes intracellular APP accumulation in primary cortical neurons”, could they please define APP. We apologize for having overlooked this abbreviation; it is now corrected. APP is the Amyloid Precursor Protein 6) In Figure 1 and 2 driver names are not very visible, could this be improved for clarity or genotypes be written in full perhaps? This has now been modified in a revised Fig.1. In Fig.2 we actually do not write the name of the driver as all experiments use repo-Gal4, as stated in the figure legend. 7)In Figure 3A, it is not very visible what the arrows is pointing at, at least not in my downloaded version of the article. This has now been modified with some zoomed-in inset to make better visible the details pointed at by the arrow." } ] }, { "id": "63653", "date": "22 Jun 2020", "name": "Jeff W. Barclay", "expertise": [ "Reviewer Expertise Invertebrate models", "genetics", "neuroscience. Referee suggested by the NC3Rs for their scientific expertise and experience in assessing 3Rs impact." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 uses Drosophila to screen by miRNAs for essential glial genes, identifying and briefly investigating one of the candidates (gartenzweg or garz). The screen appears to be conducted well, the data logically presented and the outcomes interesting and novel. The screening method and methodological algorithm is straight-forward and described well. I have no negative issues with the experiments and believe this is a valid topic for publication that will prove useful to the community. In addition, I agree whole-heartedly that the 3Rs benefits are well demonstrated for studying miRNAs, glia and GBF1 biology specifically.\nI have only minor suggestions for improvement, where the authors may want to reconsider wording to accurately reflect outcomes.\nThe overall interpretations of the manuscript are that this screen is identifying glial functions. From the manuscript, however, it is not clear what these functions actually are? The experiments manipulate expression in glial cells – and then measure broad outcome phenotypes such as lifespan and locomotion. However, I don’t think the authors are necessarily suggesting that the function of glia is lifespan or locomotion. Later there are experiments that infer potential alterations to intracellular trafficking in glia, but I find these experiments more representative of garz function in glia rather than glial function itself.\n\nThe glia are targeted by miRNA expression and RNAi in the glia themselves. As miRNAs can be potentially released by exocytosis, it would be worthwhile to discuss the possibility for effects originating in other cells. The authors have done a nice control with the RNAi in neurons; however, this has partially replicated the glial RNAi effects and thus overall may reflect some transcellular effects.\n\nThe manuscript indicates remarkable conservation in function between garz and GBF1; however, I would suggest tempering that conclusion given that they do have divergent effects (e.g. one is toxic, one is not).\n\nIt isn’t entirely clear why CD8 trafficking was selected for investigation over other possibilities. Given the lack of effect on cadherin trafficking, the speculation that overall trafficking in glia is impaired seems premature.\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? Yes", "responses": [ { "c_id": "5744", "date": "23 Jul 2020", "name": "Manolis Fanto", "role": "Author Response", "response": "We thank both reviewer for their competent assessment of our manuscript. We have now provided a revised version of our paper and reply here to the questions and the issues raised by both reviewers. 1. The overall interpretations of the manuscript are that this screen is identifying glial functions. From the manuscript, however, it is not clear what these functions actually are? The experiments manipulate expression in glial cells – and then measure broad outcome phenotypes such as lifespan and locomotion. However, I don’t think the authors are necessarily suggesting that the function of glia is lifespan or locomotion. Later there are experiments that infer potential alterations to intracellular trafficking in glia, but I find these experiments more representative of garz function in glia rather than glial function itself. We understand the point of the reviewer. To clarify, we start from the assumption that affecting glial functions (i.e. insulation, trophism, phagocytosis and neuronal activity modulation) will impact the functionality of the nervous system, hence it will affect lifespan and behaviour. This is the assumption of our screen, which indirectly addresses the functions of glias. Similarly, we find that any alterations at the subcellular level reveal a function for garz in adult glial cells, exactly as the reviewer points out. The defects in autophagy, protein localization and mitochondria are likely to underlie the defects in glial functions, but we are not focusing here on the precise way in which glial functions are affected, rather on the validation of the screen via identification of garz as an essential molecule in adult glia. 2. The glia are targeted by miRNA expression and RNAi in the glia themselves. As miRNAs can be potentially released by exocytosis, it would be worthwhile to discuss the possibility for effects originating in other cells. The authors have done a nice control with the RNAi in neurons; however, this has partially replicated the glial RNAi effects and thus overall may reflect some transcellular effects. The reviewer is correct that miRNAs can be released by exocytosis, however it is still unclear what the overall impact of this release (and uptake) is, when looking at the global organism level. While in plants and in C. elegans the transcellular effect of miRNA is well documented to be a potent effector, this appears to be much more limited in Drosophila and mammals. With respect to Garz, the simplest explanation is that this ubiquitous Golgi protein is necessary also in neurons, and, as such, it will affect lifespan also from neurons. 3. The manuscript indicates remarkable conservation in function between garz and GBF1; however, I would suggest tempering that conclusion given that they do have divergent effects (e.g. one is toxic, one is not). While we agree with this observation toxicity levels may be due to: Different expression levels. Epitope tag: GFP-Garz vs HA-GBF1. Dominant negative effect of Garz but not of GBF1. We agree that explanation 3 would be in line with some difference between GBF1 and Garz and have slightly modified our text in the discussion to account for this effect. We still think that the conservation of effect between Garz and GBF1 is remarkable in consideration of the other two possibilities and of the rescue effects. While a negative result (lack of toxicity) could be attributed to several reason, the remarkable rescue obtained by GBF1 can be explained with a high level of confidence with GBF1 performing Garz functions. 4. It isn’t entirely clear why CD8 trafficking was selected for investigation over other possibilities. Given the lack of effect on cadherin trafficking, the speculation that overall trafficking in glia is impaired seems premature. Using CD8::GFP as a general membrane reporter we aimed at checking overall intracellular membrane trafficking effects specifically within glia. CadN is expressed in many but not all glial cells and it is much more widely expressed in neurons. In this sense any defect in glial cell distribution of CadN might have been masked by the largely normal distribution of CadN in neurons. We have further clarified this point in our manuscript and apologize for not having made that clearer in our first version." } ] } ]	1	https://f1000research.com/articles/9-317
https://f1000research.com/articles/9-637/v1	23 Jun 20	{ "type": "Case Report", "title": "Case Report: Acute effect of benralizumab on asthma exacerbation without concomitant corticosteroid use", "authors": [ "Santi Nolasco", "Raffaele Campisi", "Rossella Intravaia", "Morena Porto", "Corrado Pelaia", "Nunzio Crimi", "Claudia Crimi", "Santi Nolasco", "Raffaele Campisi", "Rossella Intravaia", "Morena Porto", "Corrado Pelaia", "Nunzio Crimi" ], "abstract": "Background: Monoclonal antibodies are a relatively new therapeutic option for patients with severe refractory asthma, which can be used as an add-on to maintenance therapy, reducing the need for systemic corticosteroid usage, improving asthma symptom control and reducing exacerbations. We report a case of a patient with severe refractory eosinophilic asthma, reluctant to take systemic steroids, who was successfully treated with benralizumab alone during an acute asthma attack. Case presentation: A 59-year-old Caucasian woman with a history of allergic asthma since childhood showed a progressive decline in lung function with difficult to control symptoms and an increased number of hospitalizations despite maximal maintenance treatment, and was diagnosed with severe refractory asthma. She was reluctant to take systemic corticosteroids during exacerbations due to severe urinary retention; therefore, she started omalizumab with a partial reduction of symptoms and exacerbations over time. During a follow-up visit, she showed signs of acute exacerbation and she was switched to benralizumab during her acute phase with a rapid, dramatic amelioration of respiratory symptoms and pulmonary function, without concomitant systemic corticosteroid administration. During the treatment and at follow-up after one month, good tolerance and no side effects were observed. Conclusions: The use of benralizumab seems to be feasible, rapid, and safe in treating acute exacerbation of severe eosinophilic asthma without the use of systemic corticosteroids.", "keywords": [ "anti-IL-5 antibody", "asthma control test", "benralizumab", "eosinophilic asthma", "speed of onset", "severe refractory asthma", "asthma exacerbation" ], "content": "Abbreviations\n\nACT, Asthma Control Test; BID, bis in die; FEV1, forced expiratory volume in 1 second, FVC, forced vital capacity; FeNO, fraction of exhaled nitric oxide; LAMA, long-acting muscarinic antagonist; IV, intravenous.\n\n\nIntroduction\n\nAcute exacerbation of severe asthma is a medical emergency that requires treatment with high-dose corticosteroids and accounts for >60% of the total costs of the disease care, primarily required for emergency visits and hospitalizations1,2.\n\nMonoclonal antibodies are a relatively new therapeutic option for patients with severe refractory asthma, which can be used as an add-on to the maintenance therapy, with long-term effects such as reducing the need for systemic corticosteroids usage, improving asthma symptoms control and reducing exacerbations3. We report the use of a monoclonal antibody against interleukin-5, benralizumab, for the treatment of an acute exacerbation of severe asthma in a patient who refused to take systemic steroids.\n\n\nCase presentation\n\nWe present the case of a 59-year-old non-smoker Caucasian female, lawyer, with a history of allergic asthma since childhood.\n\nTimeline of patient's clinical evolution with diagnostic tests and treatments is shown in Figure 1.\n\nLAMA, long acting muscarinic agonist; LTRA, leukotrienes receptor antagonist; Q4W: every four weeks.\n\nThe patient had a body mass index within the normal range, chronic rhinosinusitis without nasal polyposis, and gastro-esophageal reflux under pharmacological control.\n\nHer basal asthma regimen over the years was budesonide/formoterol 160 mcg/4.5 mcg, four puffs/day, with good treatment adherence and correct inhalation technique.\n\nOver the past years, despite her adequate adherence to her maintenance regimen, the patient experienced a gradual worsening of her asthma symptoms, together with a progressive decline of her lung function and an increasing number of exacerbations, some of which required hospitalization. Therefore, after a pulmonologist consultation, combined treatment with a long-acting muscarinic antagonist (LAMA; 2.5 mcg tiotropium Respimat inhaler, two puffs daily), theophylline (300 mg tablets, bis in die) and a leukotriene receptor antagonist (montelukast, 10 mg daily) were added to her maintenance treatment regimen.\n\nIn March 2015, she was referred to our outpatient respiratory clinic at Policlinico Vittorio-Emanuele di Catania, Italy, due to the persistence of asthma symptoms despite the therapy. Pulmonary function tests showed a forced vital capacity (FVC) of 78% of predicted value (2620 mL), and a forced expiratory volume in one second (FEV1) of 55% of predicted value (1400 mL), with a post-bronchodilator (after 400 mcg of salbutamol) increase in FEV1 of 24% (1990 mL).\n\nShe showed sensitization to multiple inhalant allergens by skin prick tests (house dust mites, dog and cat dander, and Parietaria judaica), high serum total IgE levels (201 IU/mL), high blood eosinophils count (670 cells/µL) and high fraction of exhaled nitric oxide (FeNO; 51 ppb). She complained of frequent exacerbations of her asthma symptoms and recurrent hospital admissions over the past year (nearly one/month) and, therefore, we classified her as severe refractory asthma according to Global Initiative for Asthma (GINA) guidelines4.\n\nMoreover, the patient had always been reluctant to use systemic corticosteroids, referring to an almost immediate appearance of urinary retention, so she refused the proposed short course of oral corticosteroids. Therefore, we started treatment with omalizumab (300 mg via subcutaneous injections administered every four weeks).\n\nWe further evaluated our patient every four weeks, and she reported a dramatic reduction in the number and severity of exacerbations (nearly 50% less) and the number of hospitalizations overall, without using systemic steroids. Still, she described persistence of respiratory symptoms and poor asthma control with the need of a salbutamol inhaler at least five times a day, despite the maintenance and the biologic therapy. At her follow-up visit after one year from the first dose of omalizumab, her Asthma Control Test (ACT) score was 8, her FeNo was 33 ppb, and her pulmonary function tests showed an FEV1 of 73% of predicted value (1860 mL) and FVC of 90% of predicted value (3030 mL), an FEV1 / FVC of 61% and a positive post-bronchodilatation test response, with FEV1 reaching 85% (+ 12%) and 2105 mL. She underwent a chest computed tomography scan, revealing diffuse bronchiectasis; therefore, she started long-term azithromycin and airway clearance therapy on top of her usual maintenance treatment. The patient still achieved partially controlled asthma with treatment optimization.\n\nIn January 2019, she presented to the emergency department for a new severe exacerbation characterized by whistling dyspnea, greenish sputum, and acute respiratory failure, and required hospitalization.\n\nDuring the hospital stay, her sputum cytological analysis showed 17% of eosinophils out of a total count of 250,000 cells/mL, and her blood tests exhibited an elevated peripheral eosinophilic count (470 cells/µL). Pulmonary function tests revealed an FEV1 of 49% of predicted, FVC of 72% of predicted and FEV1 / FVC of 57.4%. She started intravenous (IV) theophylline (240 mg intravenous slow bolus followed by a continuous infusion of 0.5 mg/kg/h), IV piperacillin/tazobactam (4.5 g every eight hours), IV corticosteroids (40 mg of methylprednisolone), and oxygen (nasal cannula 2 liters/minute) on top of her maintenance therapy, with progressive symptoms relieved. At discharge, we proposed a switch from omalizumab to mepolizumab, but the patient refused.\n\nOn November 4, 2019, she presented for a regular outpatient visit and complained of a worsening of her usual respiratory symptoms, presence of wheezing, reduced tolerance to physical activity, abundant purulent sputum, and frequent nocturnal awakenings. She had taken 25+ puffs of salbutamol to alleviate her chest tightness. Her oxygen saturation was 91% at rest.\n\nPulmonary function tests showed an FEV1 of 61% of predicted value (1480 mL), FVC of 74% of predicted value (2410 mL), and FEV1 / FVC of 61%. A post-bronchodilatation test FEV1 reached 70% (+9%) and 1700 mL. Her ACT score was 6. She also underwent blood tests, showing elevated eosinophils (390 cells/µL) and her FeNo was 60 ppb.\n\nWe proposed a short course of steroids, but she refused to take them due to the fear of urinary retention; she also refused hospitalization. Therefore, as a rescue solution, and due to the presence of elevated eosinophils, we decided to switch omalizumab to benralizumab (30 mg by subcutaneous injection every four weeks for the first three doses, and then every eight weeks thereafter). The same day the patient started the first administration of 30 mg subcutaneous injection of benralizumab.\n\nAt a telephone follow-up after 24 hours, she reported a significant improvement in her asthma symptoms with the almost total disappearance of sputum, absence of nocturnal awakening, and noticeable reduction of dyspnea, which made her stop rescue medication. At her 48 hours follow-up visit, the physical examination revealed a dramatic decrease in whistles as compared to the day before. The FEV1 was 80% of predicted value (1940 ml, +19% compared to two days before), FVC was 88% of predicted value (2890 ml, +14% compared to two days before) and FEV1 / FVC was 67% (+6%). Oxygen saturation has improved too, reaching 98% at rest, and her blood test showed complete depletion of eosinophils in peripheral blood. After four weeks, she returned to our outpatient service for administration of the second dose of benralizumab. She described good control of respiratory symptoms within the last month with no exacerbations, nocturnal awakening, nor sputum, and only occasional use of salbutamol. She did not report any adverse effects. Her blood count continued to show complete eosinophils depletion. Pulmonary function tests showed a further increase in lung function: an FEV1 of 98% of predicted value (2360 ml), FVC of 94% of predicted value (3140 ml), and FEV1 / FVC 75%. Her ACT reached a score of 18, her highest result ever and her FeNo was 47 ppb.\n\n\nDiscussion\n\nTo the best of our knowledge, this case report is the first in the literature on the use of benralizumab administration during an acute attack of severe refractory eosinophilic asthma, without the concomitant use of systemic steroids. The main finding of this report is the efficacy and safety of benralizumab treatment during the acute phase of eosinophilic asthma exacerbation, showing a terrific and rapid response in terms of improvement of symptoms and pulmonary function (significant gain in FEV1 after only 48 hours), and reduction of sputum production, without the concomitant use of systemic corticosteroids and avoiding hospitalization.\n\nBiologics have shown long-term beneficial effects in the management of severe asthma patients5. Characterizing the properties of one molecule versus another might be crucial for a more personalized treatment approach6. The speed of treatment onset might represent an essential underrated characteristic to consider in this context.\n\nOmalizumab has been shown to reduce both asthma symptoms and exacerbations within the first 30 days of treatment7, while mepolizumab in months8 and reslizumab in weeks9. Indeed, benralizumab is responsible for rapid symptomatologic improvement, obtainable after only a few days10.\n\nThis brilliant and fast therapeutic effect is due to its antibody-dependent cell-mediated cytotoxicity activity, which results in a complete depletion of eosinophils in both peripheral blood and tissues11.\n\nThe rapid improvement in symptoms observed is in line with the results of a post-hoc analysis of two studies (SIROCCO and CALIMA), which showed positive impacts on symptoms by the third day after administration, reducing salbutamol use10. Our report strengthens the data from Nowak and coworkers12, who described similar rapid effects, indicating that the administration of one dose of benralizumab added to usual care in patients who presented with acute asthma to the emergency department decreased asthma exacerbation rate and severity as well as hospitalizations at twelve weeks.\n\nA recent case report13 described a reduction in asthma symptoms after two days and an improvement in respiratory peak flow after four days of benralizumab administration13. Rapid response to treatment was previously described in another report from our group14.\n\nThe case presented is the first in which treatment with benralizumab during the acute phase of eosinophilic asthma exacerbation without the concomitant use of systemic steroids, on top of the maintenance treatment regimen, showed rapid resolution of symptoms within 24 hours. The immediate response to the treatment is also supported by a considerable increase in pulmonary function parameters (FEV1 +19%) obtained only after 48 hours, even without the concomitant use of systemic steroids. This case could be the starting point for the use of benralizumab in the acute phase of severe eosinophilic asthma exacerbations, starting the treatment early in the emergency room without resorting to systemic steroids.\n\nThis case report has some limitations. We did not report a long-term follow-up. Therefore, we cannot document long-term beneficial effects and treatment tolerance.\n\nIn conclusion, benralizumab may represent a feasible treatment as an add-on therapy in the management of acute asthma attacks, even without the use of systemic corticosteroids. Adequately powered multicenter trials are needed to confirm our observation.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of clinical details was obtained by the patient.", "appendix": "References\n\nBahadori K, Doyle-Waters MM, Marra C, et al.: Economic burden of asthma: a systematic review. BMC Pulm Med. 2009; 9: 24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGodard P, Chanez P, Siraudin L, et al.: Costs of asthma are correlated with severity: a 1-yr prospective study. Eur Respir J. 2002; 19(1): 61–67. PubMed Abstract \| Publisher Full Text\n\nBagnasco D, Heffler E, Testino E, et al.: Pharmacokinetics and pharmacodynamics of monoclonal antibodies for asthma treatment. Expert Opin Drug Metab Toxicol. 2019; 15(2): 113–120. PubMed Abstract \| Publisher Full Text\n\nChung KF, Wenzel SE, Brozek JL, et al.: International ERS/ATS guidelines on definition, evaluation and treatment of severe asthma. Eur Respir J. 2014; 43(2): 343–373.PubMed Abstract \| Publisher Full Text\n\nMcCracken JL, Tripple JW, Calhoun WJ: Biologic therapy in the management of asthma. Curr Opin Allergy Clin Immunol. 2016; 16(4): 375–382. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSiddiqui S, Denlinger LC, Fowler SJ, et al.: Unmet Needs in Severe Asthma Subtyping and Precision Medicine Trials. Bridging Clinical and Patient Perspectives. Am J Respir Crit Care Med. 2019; 199(7): 823–829. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSorkness CA, Wildfire JJ, Calatroni A, et al.: Reassessment of omalizumab-dosing strategies and pharmacodynamics in inner-city children and adolescents. J Allergy Clin Immunol Pract. 2013; 1(2): 163–171. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaldar P, Brightling CE, Hargadon B, et al.: Mepolizumab and exacerbations of refractory eosinophilic asthma. N Engl J Med. 2009; 360(10): 973–984. PubMed Abstract \| Free Full Text\n\nCastro M, Zangrilli J, Wechsler ME, et al.: Reslizumab for inadequately controlled asthma with elevated blood eosinophil counts: results from two multicentre, parallel, double-blind, randomised, placebo-controlled, phase 3 trials. Lancet Respir Med. 2015; 3(5): 355–366. PubMed Abstract \| Publisher Full Text\n\nO'Quinn S, Xu X, Hirsch I: Daily patient-reported health status assessment improvements with benralizumab for patients with severe, uncontrolled eosinophilic asthma. J Asthma Allergy. 2019; 12: 21–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLaviolette M, Gossage DL, Gauvreau G, et al.: Effects of benralizumab on airway eosinophils in asthmatic patients with sputum eosinophilia. J Allergy Clin Immunol. 2013; 132(5): 1086–1096 e1085. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNowak RM, Parker JM, Silverman RA, et al.: A randomized trial of benralizumab, an antiinterleukin 5 receptor alpha monoclonal antibody, after acute asthma. Am J Emerg Med. 2015; 33(1): 14–20. PubMed Abstract \| Publisher Full Text\n\nIzumo T, Terada Y, Tone M, et al.: Rapid effects of benralizumab on severe asthma during surgery for residual tumor after advanced lung squamous cell carcinoma treatment with pembrolizumab. Respir Med Case Rep. 2019; 26: 292–295. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPelaia C, Busceti MT, Vatrella A, et al.: Effects of the first three doses of benralizumab on symptom control, lung function, blood eosinophils, oral corticosteroid intake, and nasal polyps in a patient with severe allergic asthma. SAGE Open Med Case Rep. 2020; 8: 2050313X20906963. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "65647", "date": "06 Jul 2020", "name": "Paula Kauppi", "expertise": [ "Reviewer Expertise Asthma", "obstructive pulmonary disease", "bronchiectasis", "allergic 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 case report of a patient who received a dose of benralizumab for acute asthma. The patient had been on omalizumab as an add on therapy with ICS, LABA, LAMA, montelukast and theophylline. She refused per oral glucocorticoids. She had moderate to severe obstruction in spirometry and bronchodilator response. She received a dose of benralizumab when having an asthma exacerbation. Two days later her asthma symptoms had decreased and lung function increased.\nWhat was adherence of the patient with her asthma medication (ICS, LABA, LAMA, montelukast and theophylline)?\n\nDid the patient smoke regularly or had she ever smoked regularly?\n\nDid the patient have pets at home?\n\nHow many exacerbations or severe exacerbations the patient had had a year before she started omalizumab?\n\nHow many exacerbations or severe exacerbations the patient had had a year before she started benralizumab?\n\nIn previous studies, benralizumab has been shown to reduce number of eosinophils in 24 hours and to increase FEV1 in 4 weeks. In this case report, benralizumab seemed to increase FEV1 in two days? Could you discuss the unexpected rapid changes?\n\nDid the patient receive some other asthma drugs or were the other asthma drugs changed on the same visit when benralizumab was started?\n\nPlease consider these two reports on use of biologicals in acute asthma: Renner A, Marth K, Schäffl-Doweik L, Pohl W. Case Report: Reslizumab in an Invasively Ventilated Patient With Acute Respiratory Failure. J Allergy Clin Immunol Pract. 2019;7(8):2922-2923. doi:10.1016/j.jaip.2019.05.0191 and Milger K, Schroeder I, Behr J, Meis T, Wulffen WV, Kneidinger N. Omalizumab Rescue Therapy for Refractory Status Asthmaticus. Ann Intern Med. 2019;170(5):351-352. doi:10.7326/L18-03592.\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": [ { "c_id": "5741", "date": "23 Jul 2020", "name": "Claudia Crimi", "role": "Author Response", "response": "Dear Dr. Kauppi, Thank you for your time and consideration in reviewing our article. Your comments were very helpful to improve the manuscript and we hope that the revised version makes it now acceptable for you. We hereby provide a point-by-point reply to your comments. We look forward hearing from you soon. Best Regards, Claudia Crimi, MD, PhD ______________________ This is a case report of a patient who received a dose of benralizumab for acute asthma. The patient had been on omalizumab as an add on therapy with ICS, LABA, LAMA, montelukast and theophylline. She refused per oral glucocorticoids. She had moderate to severe obstruction in spirometry and bronchodilator response. She received a dose of benralizumab when having an asthma exacerbation. Two days later her asthma symptoms had decreased and lung function increased. 1. What was adherence of the patient with her asthma medication (ICS, LABA, LAMA, montelukast and theophylline)? Reply:Thank you for your comment. Based on your suggestion we have added a sentence stating that the patient’s adherence to both inhaled (ICS + LABA and LAMA) and oral (theophylline and montelukast) therapy continues to be very good, due to her fear of possible need for intravenous/oral steroids in case of non-adequate adherence (see the section “Patient information and medical history” last sentence). 2. Did the patient smoke regularly or had she ever smoked regularly? Reply:Thanks for pointing out this. No, the patient has never smoked; this information is present in the first sentence of the paragraph “Patient information and medical history”. 3. Did the patient have pets at home? Reply:Thanks for this comment. No, the patient has never had pets at home; we have now clarified this in the description of patient’s medical history (Second sentence of the paragraph “Patient information and medical history”). 4. How many exacerbations or severe exacerbations the patient had had a year before she started omalizumab? Reply:Thanks for highlighting this aspect. As stated in the section “Initial presentation, diagnostic tests and treatment” (please see second paragraph) the patient complained one exacerbation per month (12 exacerbations/year) over the past year before staring omalizumab treatment. 5. How many exacerbations or severe exacerbations the patient had had a year before she started benralizumab? Reply:Thanks for highlighting this aspect. As stated in the section “Subsequent presentation, diagnostic tests and treatments” (please see first sentence) the patient reported a reduction of nearly 50% of exacerbation after one year of omalizumab (one exacerbation every 2 months - 6 exacerbations/year) before staring benralizumab treatment. 6. In previous studies, benralizumab has been shown to reduce number of eosinophils in 24 hours and to increase FEV1 in 4 weeks. In this case report, benralizumab seemed to increase FEV1 in two days? Could you discuss the unexpected rapid changes? Reply:The patient showed a complete depletion in peripheral blood eosinophil count within 2 days after switching from omalizumab to benralizumab, showing an increase of pre-bronchodilator FEV1% (+19%), FVC (+14%) and FEV1/FVC (6%). This rapid eosinophils reduction is consistent with previous benralizumab studies [1] and reflects its mechanism. Moreover, benralizumab has shown to significantly reduce eosinophils not only in peripheral blood but also in airways, limiting the release of noxious eosinophil granule proteins in bronchial tissues [2,3]. IL-5 is upstream of IgE in the canonical allergic inflammatory cascade, coupled with current emerging evidence of non-IgE-mediated alternative IL-5 pathways that can maintain eosinophilia is reasonable to expect an anti-IL-5R mAb to be effective when an anti-IgE mAb fails to curb symptoms [3]. We suggest that these mechanisms have been responsible for the rapid improvement of the patient's lung function. In our patient, eosinophils reduction allowed the tapering of type-2 inflammatory processes accountable for her asthma exacerbations. These considerations are corroborated by the good control of respiratory symptoms, and further respiratory function improvements 4 weeks after the first benralizumab administration. Therefore, our observation confirms that the therapeutic responses to anti-asthma biological drugs are strictly individual, being dependent on each patient's specific endotype. We have discussed this point in the discussion section, as requested. Nowak RM, Parker JM, Silverman RA, et al. A randomized trial of benralizumab, an anti interleukin 5 receptor αmonoclonal antibody, after acute asthma. Am J Emerg Med. 2015;33(1):14-20. doi:10.1016/j.ajem.2014.09.036 (REF 13) Laviolette M, Gossage DL, Gauvreau G, et al.: Effects of benralizumab on airway eosinophils in asthmatic patients with sputum eosinophilia. J Allergy Clin Immunol. 2013; 132(5): 1086–1096 e1085 (REF 12) Mukherjee M, Bakakos P, Loukides S. New paradigm in asthma management: Switching between biologics!. Allergy. 2020;75(4):743-745. doi:10.1111/all.14038 (REF 16) 7. Did the patient receive some other asthma drugs or were the other asthma drugs changed on the same visit when benralizumab was started? Reply:Thank you for highlighting this aspect. No. The patient at this point was already taking high-dose ICS+LABA, LAMA, montelukast, theophylline, omalizumab (last administration exactly 4 weeks prior), azithromycin and 25+ daily puffs of salbutamol. During the visit, due to presence of wheezing, reduced tolerance to physical activity, abundant purulent sputum, frequent nocturnal awakenings and rest oxygen saturation of 91%, her reluctance to be hospitalized and to take oral or systemic corticosteroids, and because of the presence of 390 eosinophils/µL we decided to immediately switch from omalizumab to benralizumab 30mg. We have now clarified this aspect adding a sentence at the end of the section “Subsequent presentations, diagnostic tests and treatments” (second-last sentence). 8. Please consider these two reports on use of biologicals in acute asthma: Renner A, Marth K, Schäffl-Doweik L, Pohl W. Case Report: Reslizumab in an Invasively Ventilated Patient With Acute Respiratory Failure. J Allergy Clin Immunol Pract. 2019;7(8):2922-2923. doi:10.1016/j.jaip.2019.05.0191and Milger K, Schroeder I, Behr J, Meis T, Wulffen WV, Kneidinger N. Omalizumab Rescue Therapy for Refractory Status Asthmaticus. Ann Intern Med. 2019;170(5):351-352. doi:10.7326/L18-03592. Reply:Thank you for referring to these interesting references. Both reports showed the rapid effects of biologics, but, as stated by the authors, it is not possible to exclude that patients would have improved anyway due to the high doses of systemic bronchodilators and corticosteroids not possible to establish the effect of biologic alone. Our case is the first report of an acute asthma exacerbation treated with benralizumab without the addition of systemic corticosteroid, antibiotic or aggressive bronchodilator therapy, being benralizumab administered on top of patient’s maintenance treatment. Therefore, in our report, it is likely to verify and measure the real effect of benralizumab alone during an acute asthma exacerbation, with complete eosinophils reduction and pulmonary function improvement only after 48 hours benralizumab 30 mg subcutaneous administration. We have now discussed the references as suggested." } ] }, { "id": "65420", "date": "08 Jul 2020", "name": "Claudio Micheletto", "expertise": [ "Reviewer Expertise severe asthma" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe Authors presented a case report on the use of benralizumab administration during an acute attack of severe refractory eosinophilic asthma, without the concomitant use of systemic steroids. Benralizumab is an anti-receptor monoclonal anti-IL-5 antibody, which has been shown to be particularly effective in reducing exacerbations, used as regular therapy in severe eosinophilic asthma.\nSome questions:\nHow was the patient's adherence to inhalation therapy?\n\nDid the patient have a previous accurate diagnosis of severe asthma?\n\nWas the patient then regularly treated with benralizumab?\n\nWere eosinophils measured in the following weeks?\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": "5740", "date": "23 Jul 2020", "name": "Claudia Crimi", "role": "Author Response", "response": "Dear Dr. Micheletto, Thank you for your time and consideration in reviewing our article. We are glad that you find our case report interesting. We were thrilled to read the approval from an expert in the field like you. We have now added a bit more information as you requested. Best Regards, Claudia Crimi, MD, PhD ___________________________ Specific point-by-point response: The Authors presented a case report on the use of benralizumab administration during an acute attack of severe refractory eosinophilic asthma, without the concomitant use of systemic steroids. Benralizumab is an anti-receptor monoclonal anti-IL-5 antibody, which has been shown to be particularly effective in reducing exacerbations, used as regular therapy in severe eosinophilic asthma. · How was the patient's adherence to inhalation therapy? Reply:Thank you for your comment. Based on your suggestion we have added a sentence stating that patient's adherence to both inhaled (ICS + LABA and LAMA) and oral (theophylline and Montelukast) therapy, continue to be very good, also due to her fear of possible need for intravenous/oral steroids in case of non-adequate adherence. · Did the patient have a previous accurate diagnosis of severe asthma? Reply:Thank you for your comment. We diagnosed severe asthma according to ERS/ATS guidelines [1]; therefore, we excluded other respiratory diseases that may share common clinical manifestations of severe asthma (i.e., bronchopulmonary aspergillosis, vasculitis, chronic cough). We have now specified it in the text and added the appropriate reference as below. Chung KF, Wenzel SE, Brozek JL, et al.: International ERS/ATS guidelines on definition, evaluation and treatment of severe asthma. Eur Respir J. 2014; 43(2): 343–373. · Was the patient then regularly treated with benralizumab? Reply: Thank you for pointing out this aspect. Yes, the patient was then treated regularly with benralizumab 30 mg subcutaneously every four weeks for the first three doses and then every eight weeks. We have better clarified this in the text (paragraph follow-up and outcomes, last sentence). · Were eosinophils measured in the following weeks? Reply:Thank you for highlighting this aspect. Yes, we executed blood eosinophils count 48h and exactly four weeks after the first benralizumab administration (right before the second dose), showing a complete eosinophils depletion in both determinations (as described in the paragraph follow-up and outcomes). Moreover, a blood test was performed before every next benralizumab dose. We have now better specify this aspect in the text (paragraph follow-up and outcomes, last sentence)." } ] } ]	1	https://f1000research.com/articles/9-637
https://f1000research.com/articles/8-1770/v1	17 Oct 19	{ "type": "Research Article", "title": "CCL2 and IL18 expressions may associate with the anti-proliferative effect of noncontact electro capacitive cancer therapy in vivo", "authors": [ "Rarastoeti Pratiwi", "Nyoman Yudi Antara", "Lalu Gunawan Fadliansyah", "Syamsul Arif Ardiansyah", "Luthfi Nurhidayat", "Eti Nurwening Sholikhah", "Sunarti Sunarti", "Sitarina Widyarini", "Ahmad Ghitha Fadhlurrahman", "Hindana Fatmasari", "Woro Anindito Sri Tunjung", "Sofia Mubarika Haryana", "Firman Alamsyah", "Warsito Purwo Taruno", "Nyoman Yudi Antara", "Lalu Gunawan Fadliansyah", "Syamsul Arif Ardiansyah", "Luthfi Nurhidayat", "Eti Nurwening Sholikhah", "Sunarti Sunarti", "Sitarina Widyarini", "Ahmad Ghitha Fadhlurrahman", "Hindana Fatmasari", "Woro Anindito Sri Tunjung", "Sofia Mubarika Haryana", "Firman Alamsyah", "Warsito Purwo Taruno" ], "abstract": "Background: Noncontact Electro Capacitive Cancer Therapy (ECCT) is a novel treatment modality in cancer. Chemokine (C-C motif) ligand 2 (CCL2) has a major role in the outgrowth of metastatic breast cancer. Interleukin 18 (IL18) plays a role in macrophage alteration, which leads to excessive angiogenesis. This study aims to elaborate on the association of CCL2, IL18, IL23α, and TNF-α (tumor necrosis factor-alpha) expression with the anti-proliferative effect of ECCT in rat breast tumor tissue.\n\nMethods: Low intensity (18 Vpp) and intermediate frequency (150 kHz) alternating current-electric field (AC-EF) between two capacitive electrodes were exposed as external EF to a rat cage. Twenty-four rats were divided into four groups of six replicates. Breast tumor tissues were collected from 7, 12-dimethylbenz[a]anthracene (DMBA)-induced rats. Two groups were none DMBA-induced rats without ECCT exposure (NINT) and with (NIT). The other two groups were DMBA-induced rats without ECCT exposure (INT) and with (IT). Mammary glands and breast tumor tissues were collected from each group and preserved. Hematoxylin-eosin and immunohistochemistry staining were performed on paraffin sections of tissues using anti-PCNA, anti-ErbB2, anti-Caspase3, and anti-CD68. CCL2, IL18, IL23α, and TNF-α mRNA relative expressions were analyzed using qRT-PCR. Results: ECCT exposure may cause the reduction of PCNA protein expression as well as ErbB2 on breast tumor tissues, but it causes the increase of Caspase3 and macrophage CD68 protein. In rat breast tumor tissues of IT groups, the mRNA expression of CCL2 and IL18 are significantly down-regulated, in contrast with the up-regulated expression of these cytokines in tumor tissues of the INT group. IL23α and TNF- α expression remained similar in both groups. Conclusion: CCL2 and IL18 expressions have an association with the inhibition of breast tumor cell proliferation affected by ECCT exposure", "keywords": [ "ECCT", "rat breast tumor", "anti-proliferative", "IL18", "CCL2" ], "content": "Introduction\n\nElectro Capacitive Cancer Therapy (ECCT) has been developed as a noncontact alternating current (AC) electric field (EF)-based cancer therapy method. A previous study1 reported that ECCT low intensity (18 peak-to-peak voltage) intermediate frequency (100 kHz) treatment inhibits the growth of MCF-7 cells. Furthermore, this EF therapy could reduce the tumor size of C3H mice-breast tumor model, without abnormality in the dermal tissue and mammary glands of sham mice1. Moreover, Mujib et al.2 suggested that ECCT exposure (100–200 kHz) might induce p53 expression in cancer cells, such as oral squamous cell carcinoma, HeLa, and bone marrow mesenchyme cells. A previous study, using Tumor Treating Electric Field (TTFields) demonstrated that AC-EF with low intensity and intermediate frequency exposure to tumor cells caused the failure of tumor cell division toward the mitotic phase3,4. A following study reported that the failure of tumor cell division was due to the disruption of spindle microtubule assembly, but not in normal cells5. From those studies, it was suggested that the failure of tumor cell division activates tumor cell apoptosis. However, the evidence that AC-EF exposure disturbs cancer cell proliferation and the molecular mechanism underlining this cell disturbance and elimination remains unknown.\n\nSolid tumor growth and development are dependent on inflammatory cells in its microenvironment. Stromal components, such as endothelial cells, myeloid derivate suppressor cells and macrophages, reside in the solid tumor microenvironment6. Macrophages and tumor cells act as inflammatory cells that can be affected by chemical signals, i.e. cytokines and chemokines7,8, and electric fields9. A previous study reported that CCL2 is a chemo-attractant that binds to its receptor (CCR2) on monocytes, macrophages, and lymphocytes. In a previous in vivo study, CCL2-induced chemokine cascade in macrophage-associated metastasis (MAM) produced another ligand, CCL3, for metastatic seeding of breast cancer cells7,10 In addition, IL18 plays a role in the migration of breast cancer cells via down-regulation of claudin12 and p38 MAPK (mitogen activation kinase) pathway11. Hoare et al.9 demonstrated that electrical signals have been identified as major contributors to the coordination and regulation of macrophage functions. So far, electric fields-based therapies need to be described in order to understand the modulation of macrophage function, which underline the solid tumor microenvironment.\n\nTumor necrosis factor-alpha (TNF)-α cytokine is one member of the tumor necrosis factor superfamily with a wide spectrum of biological activity. Meneggati et al.12 reviewed TNF-α early on, and this inflammatory cytokine was suggested to be a potential antitumor agent and inducer of apoptosis in vitro. However, in the following years, recent studies reported that TNF-α significantly induces breast cancer metastasis via TNFα-activated mesenchyme stem cells (MSCs) in a lung metastasis model of murine breast cancer13–15. Although many studies focus on the function of TNF-α in the solid tumor microenvironment, the function remains as yet not fully clarified. So far, we understand that macrophages are multifunctional in the solid tumor microenvironment. Tumor-associated macrophages (TAMs) help tumor cell growth by releasing several pro-inflammatory cytokines, such as TNFα and IL23, which are expressed by the classically activated M1 macrophage16. A previous study suggested that IL-23α is involved in inflammation and angiogenesis activities in the tumor microenvironment in spite of moderating CD8+ T-cell infiltration17. Furthermore, evaluation of IL-23 suggested that this cytokine has a function in promoting tumor metastasis and growth by up regulating matrix metalloproteinase (MMP)-918. On the other hand, Zimolag et al.19 reported that direct current (DC)-EF in the physiological condition might reposition MCSs into a wound site and allow macrophages to be at short distance to the wound. Therefore, the expression of either TNFα or IL-23α cytokines-produced inflammatory cells in the microenvironment of solid breast tumor during the physiological DC-EF or AC-EF need to be further examined.\n\nThe anti-proliferative effect of AC-EF has been reported using several cell lines and in some tumor animal models. As reported by Ma et al.20, DMBA-induced breast cancer enhanced chromosomal instability and increased ErbB2-mediated mammary carcinogenesis. However, the association and mechanism of killing tumor cells and how the immune system clears death cells needs to be further clarified. A recent study demonstrated that TTFields therapy activates macrophage specific immune responses through the activation of several cytokines, such as NF-kB (nuclear factor-kB), TNF-α, and IL1-β in vitro10. In the present study, we elaborate on the relationship between the anti-proliferative effect of ECCT on DMBA induced-rat breast cancer cell growth and on the activity of inflammatory cells to express cytokines IL18, TNF-a, IL23 and chemokine CCL2, which play an essential role to the development of solid breast tumors. The use of this breast tumor animal model in this study is needed, because this model can represent the condition of breast cancer in patients. The main objective of this study is to examine the effectiveness of ECCT treatment on tumor growth inhibition through the molecular communication among stromal cells in the solid microenvironment tumor.\n\n\nMethods\n\nThis study was carried out at the animal house of LPPT Research Center Universitas Gadjah Mada (UGM) which is accredited by ISO/IEC 17025:2000 (a laboratory management standard). All requirements for animal welfare following the LPPT Ethics Committee Guidance have been fulfilled. This animal experiment has been legalized with an Ethical Clearance certificate number: 00029/04/LPPT/2018. The rat number for experimental design was calculated for the minimal biological replication of rats, with four treatment groups according to the Federer Formula21. 24 female rats (Rattus norvegicus Berkenhout, 1769) Sprague Dawley (SD) strain, five weeks old and weighing 50–80 grams were used in this study. The rats ware obtained from LPPT Research Center.\n\nRats were fed with AIN-93M standard diet and standard water ad libitum. Rats were placed in a standard animal room (temperature and humidity were 20 to 25 °C and 40 to 60%, respectively). Rats were acclimatized to the laboratory condition and standard cage for 5 days.\n\nThis study focused on samples of rat breast tumor and healthy mammary gland. Therefore, we only observed the minimal number of tissue samples required for replication; three tissues samples per treatment group (4 groups) was used for biological replication. Each sample was measured twice for qRT-PCR analysis and triple sections per tissue sample for histopathologic scoring requirements. Each rat was marked individually using picric acid (non-toxic) staining. Rats were observed for behavior, general physical conditions such as hairs, eyes, noose, ears, and feces every day. Rats were weighed every three days during the experiments using the balance scale for rodents. Rat welfare was maintained following the standard protocol from LPPT Research Center.\n\nThe experimental design of ECCT exposure treatments used four rat gr6oups, six biological replicates, which consisted of:\n\n- NINT group: non-DMBA-induced rats, non-therapy exposure (n=3)\n\n- NIT group: non-DMBA-induced rats, therapy exposure (n=3)\n\n- INT: DMBA-induced rats, non-therapy treatment (n=3)\n\n- IT: DMBA-induced rate, therapy exposure (n=3)\n\nRats were induced ten times with DMBA22,23 doses of 20 mg/kg body weight within five weeks by oral administration. The DMBA (Sigma Aldrich; cat. no. D3254-1G) administrations were done around 04 pm in the animal room by the technician of LPPT Research Center following the Standard Operational Procedure (SOP) for DMBA treatment animal. After DMBA administration, all rats were palpated every two days using the standard procedure of palpation from LPPT Research Center. The tumor nodule was observed around 4 to 6 weeks after DMBA administration. Tumor nodule diameters were measured every 2 days using digital caliper (Fisher Scientific) and all data measurements were tabulated.\n\nSolid tumor (± 1 cm tumor size) bearing rats were exposed to an AC-EF of 150 kHz and low intensity of 18 Vpp. Therapy was performed for 21 days, with a total exposure of 10 hours per day with 2 hours rest. The starting time of ECCT treatments were at 06 to 11 am, then at 01 to 06 pm. During ECCT-exposing in the individual ECCT cage (designed by Ctech Labs Edwar Teknologi, IDN Patent REG. P00201200011), rats were fed with cucumber ad libitum, however during rest hours, rats were fed with a standard diet and water ad libitum in a communal cage with standard bedding and feeding for 5 rats. The ECCT treatment was finished after 21 days of treatment. Rats were sacrificed (euthanasia) by ketamine hydrochloride (KETALAR® Pfizer; cat. no. 629-24006) injection with a dosage of 150mg/kg body weight on the day after the last treatment. Rats were sacrificed starting at around 08 am with the standard ethics procedure for rat euthanasia and surgery. After taking the samples, the remaining dead rat bodies were put in the freezer prior to eradication of the carcinogenic (DMBA) contaminated animals using the SOP of the LPPT animal house. Mammary glands and solid tumor tissues were sliced and fixed in 10% NBF (neutral buffer formalin, Bio-Optica; cat. no. 05-K01004) with ratio 5:1 for histological examination and in RNAlater® (Invitrogen; cat. no. AM7024) solution for total RNA extraction.\n\nMammary glands and solid tumor tissues were fixed in NBF and then processed using the paraffin method and stained with hematoxylin-eosin using the procedures provided by Bancroft and Cook24. Summarily, the samples were periodically washed with 70% alcohol and subsequently dehydrated using a higher concentration of alcohol (80–100%). The dehydrated samples were then cleared with toluene (Merck; cat. no. 1083252500) overnight. The samples were infiltrated with paraffin (Merck; cat. no. 1073372500) in a 65°C oven and then embedded with freshly prepared paraffin. The sample paraffin blocks were sectioned with microtome (Microm HM 315) providing a 4–6 um thick slice, which were then placed on a slide. Later on, the samples were then deparaffinized using xylene (Merck; cat. no. 1086612500), rehydrated using a downgraded concentration of alcohol (96–40%), and finally stained with Hematoxylin (made from Hematoxylin Krist C.I.75290, Merck; cat. no. 1159380025, using Erlich’s formulation) and Eosin solution (made from Eosin Y, CI. 45380, Merck; cat. no. 1159350025). The stained samples were subsequently dehydrated using upgraded level of alcohol, cleared in xylene and, lastly, mounted with Entellan (Merck; cat. no. 1079600500) and coverslip. Histology slides were observed under Leica Scanscope AT2 at 0.5 µm/pixel resolution with 50 fields of view.\n\nThe 4–6 um thick paraffin section of samples were placed on a Poly-L-lysine coated slide. The INT and IT tumor tissues samples were then processed using the Starr Trek Universal-HRP Detection Kit (Biocare Medical; cat.no BRR 700 AH, AL10) using the manufacturer’s protocols. In brief, the samples were deparaffinized using xylene and then rehydrated with downgraded concentration of alcohol. The samples were heated in the microwave with sodium citrate buffer pH 6.0 for antigen retrieval for 15 minutes at 95 °C. The samples were soaked with 3% H2O2 (Sigma-Aldrich) in PBS for 5 min to block endogenous peroxidase and subsequently treated with Background Sniper for 20 minutes for suppressing nonspecific binding. Afterwards, samples were separately incubated with anti-PCNA (ABCAM; cat.no. ab18197), anti-caspase-3 (ABCAM; cat.no. ab13847), anti-CD68 (ABCAM; cat.no. ab201340), and anti-ErbB 2 (ABCAM; cat.no. ab16901) antibodies overnight at 4 °C, followed by Trekkie Universal Link incubation for 60 minutes. Then, the samples were incubated with Trek-avidin HRP Label for staining development and then counterstained with hematoxylin. Lastly, the samples were dehydrated using an upgraded concentration of alcohol, cleared with xylene, and mounted with Entellan and coverslip. Immunohistochemistry slides were observed under Leica ICC50 E at 0.5 µm/pixel resolution with 50 fields of view.\n\nQuantitative-RT-PCR (qRT-PCR) was applied for measuring the transcriptomic expression (mRNA) of IL18, CCL2, TNF-α, and IL23α genes. Isolation of RNA was performed using Total RNA Mini Kit (Blood/cultured cell; Geneaid; cat.no RB100), and cDNA synthesis with Superscript® III first-strand synthesis supermix (Invitrogen; cat.no 18080-400). Primers were as follows:\n\n- IL23α F: CAGGTTCCCATGGCTACAGT, R: TCTGGGGTTTGTTGCTTTTC\n\n- IL1825 F: CAGACCACTTTGGCAGACTTCA, R: ACACAGGCGGGTTTCTTTTGT\n\n- TNF-α25 F: AGCATGATCCGAGATGTGGAA, R: AATGAGAAGAGGCTGAGGCACA\n\n- CCL226 F: 5’ GTGCTGTCTCAGCCAGATGCAGTT 3’, R: 5’ AGTTCTCCAGCCGACTCATTGGG 3’.\n\n- GADPH27 F: 5’ TGACAACTTTGGCATCGTGG 3’, R: 5’ GGGCCATCCACAGTCTTCTG 3’\n\nRT-PCR analysis was performed using Universal SYBR® Green Supermix paint SsoAdvanced (BIO-RAD; kit. No. 172-5270). The thermal cycling conditions for amplification of IL18, TNFα, IL23α, CCL2 and GADPH were the same for pre-denaturation at 95°C, 30”; and denaturation at 95°C, 10”. However, the annealing conditions were different: IL18 TNFα, and GADPH, 60°C for 10”; IL23, 60°C for 15”; and CCL2, 65°C for 10”. All RT-PCR reactions were done using a BIO-RAD CFX96 TM Real-Time System, C1000 Touch®Thermal Cycler machine. qRT-PCR data was calculated using the Livak method28.\n\nData analysis for IHC was scored using ImageJ version 1.51 software29. The independent-T test with IBM SPSS Statistics v22 was used for image scoring between groups and for qPCR data analysis. All graphs in this article were created by GraphPrism 7 software.\n\n\nResults\n\nThe rats prior and during ECCT treatment did not have pathogen infection, as observed during daily observation. During the experiments, the rats’ body weight for all groups was not significantly different with the rat base line or untreated rats (secondary data not shown). Results of ECCT-exposed rat breast tumor (IT) show that the increase of the fluid bathing the tumor may affect the tumors size. This is in contrast with the results from the sham rat breast tumor group (INT), which exhibited denser tissues inside the solid tumor and slower growth (Figure 1).\n\n(A) Representation of solid tumor mass after surgery and (B) the solid breast tumor growth curve based on the diameter of tumor nodules. Tumor growth measurements were taken every two days for 21 days. INT= DMBA-induced rats without therapy, IT= DMBA-induced rats with therapy exposure.\n\nBased on histological sections using hematoxylin-eosin staining (Figure 2), the observation of ECCT exposure effect on average rat mammary glands shows that there is no morphological tissue damage in both treatments, NINT nor NIT rat groups (Figure 2A and B). In solid tumor tissue sections, DMBA-induced tumor cells grew massively in both treatments (Figure 2C, D, E and F). Decreasing of fat and myoepithelial cells, and increasing of necrotic cells in mammary tissues due to the high tumor cell proliferation activity and minimal blood supply for healthy cells30. The lumens of mammary glands were filled with massive proliferative cells (Figure 2 C, D, E and F), in contrast with the healthy mammary gland which contain a layer of epithelial cells (Figure 2 A and B). According to Denisov et al.31, breast tumors develop morphological diversity related to the tumor progression. The morphological tumor cell growth pattern, such as solid tumor, reveals tens to hundreds of groups of shapeless tumor cells. This solid tumor cell pattern is related to tumor invasion or bad prognosis. The tubular structures or tube-shaped pattern of tumor growth is more similar with normal tubular mammary ducts31. Figure 2 C and E (INT group) demonstrated a more solid tumor and less tubular tumor pattern. In contrast, the tubular tumor structure was observed more frequently in the IT group (Figure D and F) than in INT group. In general, tumor growth patterns of both INT and IT groups have solid tumor morphology. Therefore, those tumor can be identified as breast adenocarcinoma31. The breast tumor cells grow invasively to other healthy tissues and caused necrosis area on both treatments, since the healthy myoepithelial and endothelial cells of blood vessels were sited on the solid tumor sections. However, other tissues were removed from the observed-tumor tissue. On the sections of ECCT-treatment (IT), the invasive tumor cells showed a reduced number of mitotic cells and more apoptotic cells (Figure 2E and F). Moreover, it can be seen that lumen epithelial cells were more frequent for ECCT-exposure treatment than in tumor tissues without ECCT-exposure.\n\n(A and B) There is no morphological change both on mammary gland tissue of none DMBA-induced rat non-therapy (NINT; A), and with therapy (NIT; B). Hematoxylin and eosin staining, magnification 100x. (C and D) In contrast, adenocarcinoma breast tissue of DMBA induced rat nontherapy (INT; C) shows more massive tumor cells and fewer lumens than the tumor section with therapy (IT; D). Hematoxylin and eosin staining, magnification 400x. (E and F) Mitotic figure (black arrow) and apoptotic figure (yellow arrow) on breast tumor tissues of INT and IT group, respectively. FC= Fat Cells, L=Lumens, EC=Epithelial Cells, M= Myoepithelial Cell, NA= Necrotic Area, BV= Blood Vessels.\n\nFigure 3 shows the appearance of the protein of PCNA, ErbB2, Caspase 3, and macrophage CD68 on solid tumor tissues of ECCT treatment either with ECCT (IT) or without (INT). The percentage of tumor cells expressing PCNA protein in the IT group was significantly lower than in the INT group (p< 0.01). Consistent with PCNA expression, ERBB2 protein expression shows a significant decrease in tumor tissues of the IT group compared to the INT group (p<0.05). In contrast, the appearance of Caspase 3 and CD68 proteins on tumor tissues of the IT group was significantly higher than the INT group (p<0.01).\n\n(A and B) Anti-PCNA, (C and D) anti-ErbB2, (E and F) anti-Caspase-3, (G and H) anti-CD68. Percentage of positive cells of (I) PCNA, (J) ErbB2, and (K) Caspase-3 in 50 fields of view. (L) Count of total macrophages in 50 fields of view. Observation of histological slide was performed using Leica CC50 E at 0.5 µm/pixel resolution at 400x. Bar=100 µm. The mean, standard deviation of the data experiment show , p<0.05, , p<0.01. INT= DMBA-induced rats without therapy, IT= DMBA-induced rats with therapy exposure.\n\nThe relative mRNA expression of CCL2, IL18, TNF-α, and IL23α genes can be seen in Figure 4. The appearance of CCL2 was significantly lower on solid tumor tissues of the IT group than the INT group (p<0.01). This result was consistent with decreasing IL18 expression on tumor tissues of IT group compared to the INT group. In contrast, the results of TNF-α and IL23α expression in both groups was not significantly different. However, TNF-α gene expression was up-regulated relatively and IL23α gene expression was down-regulated relatively with the internal control of GADPH gene expression. These results suggest that mRNA relative expression of CCL2 and IL18 were more down-regulated in tumor tissues exposed-ECCT (IT group) in comparison with the INT group. Whereas on the solid tumor tissues, the relative mRNA expression of both TNF-α and IL23α genes were similar.\n\nAmplification and melt peak chart of (A and B) CCL2, (C and D) IL18, (E and F) IL23α and (G and H) TNF-α. Relative expression of (I) CCL2, (J) IL-18, (K) IL23α and (L) TNF-α genes with NINT as a control group. The internal control of relative gene expression is GADPH gene. (M) Relative expression of TNF-α, IL23α, IL-18, and CCL2 INT vs. IT. Error bar shows a standard deviation of three replications; *p<0.01.\n\n\nDiscussion\n\nDMBA-induced rat solid breast tumor has been studied in this study. Tumor interstitial fluid (TIF) formation could be affected by ECCT treatment, while other solid tumors not exposed to ECCT did not show a clearly visible effect. Although the substantial tumor size after ECCT exposure was not reduced, the solid tumor texture became soft and fluid (Figure 1A and B). According to Wagner and Wig32, the TIF contains stromal and immune cells able to produce inflammatory substances into the solid tumor environment. TIF is an essential source of tumor-specific proteins that can be used to examine the effect of therapy on the mechanism of tumor-promotion or inhibition. Here we connected the TIF phenomenon and the growth of tumor cells after treating rats with AC-electrical fields exposed ECCT. A previous study5 using TTField (200 kHz) reported that this electric field treatment could influence not only tumor mitotic arrest and cell death, but also on cellular movement, infiltration and migration using in vitro and in vivo tests.\n\nOne of the critical modalities in cancer therapy is to minimize side effects resulting from the treatment. Our results (Figure 2A and B) demonstrated that ECCT exposure on non-DMBA induced rats showed healthy mammary gland tissue, similar to those not exposed to ECCT (NIT and NINT groups, respectively). Normal lobules consisted of an epithelial cell layer in both treatments and there was no tissue damage, such as an atrophic duct or other tissue injuries. In the rat solid tumor tissues we observed an abundance of proliferative cells that expanded to most areas of mammary glands. However, the condition of solid tumors treated with ECCT was better with than without exposure (Figure 2C and D). Tumors treated with ECCT had a lower number of mitotic figures than the sham tumor treatment. Indeed, the apoptotic figures on tumor tissues with therapy were higher than non-therapy tissues (Figure 2E and F). Necrotic tissue areas were found in both treatments, but were more frequent in ECCT exposed rat breast tumor tissues than non-exposed. A previous study on tumor cells and its microenvironment interaction suggested that molecules and intermediate signaling substances play a role in controlling cell infiltration, survival, and apoptosis, which occur at the same time. So far, these molecules could be used as molecular targets for anti-cancer therapy modality33.\n\nAlamsyah et al.1 reported a decrease in solid tumor size after exposure with 100 kHz AC-EF ECCT. But in this study, we obtain different results, where the tumor size did not decrease using 150 kHz with a similar treatment method. Indeed, tumor size was not significantly increased. ECCT and TTFields are noninvasive cancer treatment modalities based on AC-EF with an intermediate frequency (100–200 kHz) and low intensity, however, they differ in noncontact and direct contact on skin, respectively, and wave type1,3. A recent study10 reported that TTFields influence the activation of macrophage-specific immune responses through apoptotic bodies of dead tumor cells due to the agitation of cell mitosis by EF. These mechanisms should be further examined using molecular approaches, e.g. transcriptomics, to target proteins specifically affected by EF based cancer therapy.\n\nProtein PCNA is frequently used as a cell proliferation marker. In this study, we evaluated the effect of ECCT treatment as an anti-proliferative agent using an intermediate frequency of 150 kHz. Previous studies examined the anti-proliferative effects using several types of cancer cell lines and measurement of tumor size in vivo1,2. In the current study, anti-proliferative activity can be seen in Figure 3A, B and I, which revealed that ECCT exposure enables inhibition of breast tumor cell proliferation (p<0.001). This result is consistent with previous findings1,2. In addition, Giladi et al.5 exposed TTFields 150 kHz on non-small cell lung cancer cell lines and KLN205 squamous cell carcinoma in mice, which revealed inhibition of cancer cell viability and lung robust tumor growth due to the effect of AC-EF (TTFields) treatment. According to Ma et al.20, DMBA promotes ErbB2-mediated carcinogenesis via genomic instability. In the present study (Figure 3C, D and J), DMBA induced rats bearing solid breast tumors, which were exposed with ECCT treatment (IT), a significantly lower percentage of ErbB2 expressed cells were observed compared to the non-ECCT treatment (INT). Therefore, the present study supports previous results that suggested the anti-proliferative effect of AC-EF based tumor or cancer therapy (TTField and ECCT).\n\nThe most crucial evidence of cancer therapy is reducing tumor cell growth caused by cell death. Apoptosis is a necessary cell death process for inhibiting metastatic cancer34. The present study shows that the percentage of breast tumor cells expressing Caspase 3 (effector caspase) in the IT group is higher than in the INT group (Figure 3E, F and K). It can be suggested that ECCT exposure disturbs cell mitosis toward cell death through apoptosis, autophagy or necrosis. However, this result indicates that apoptosis via caspase 3 might be a predominant mechanism of breast tumor cell death caused by ECCT-treatment. This result is consistent with the effect of reducing cell proliferation (using PCNA cell proliferation marker) in the IT group. Mujib et al.2 suggested that p53 might be involved in apoptosis induction in several cancer cell lines via a caspase-dependent apoptosis mechanism after exposure to ECCT. The results shown by the present study need to be confirmed with other protein markers involved in the tumor cell death mechanism.\n\nThe clearance of cell death debris is the final stage of apoptosis. In this process, it involves phagocytic cells, such as macrophages. The current study (Figure 3 G, H, and L) showed increasing expression of CD68 (a classical macrophage marker) on breast tumors exposed to ECCT (IT group), which was significantly higher than non-therapy (INT). Macrophages are multifunctional in solid tumor microenvironments, including macrophage polarization M1 and M2 which are involved in TAM and MAM functional directions7,35. Ni et al.36 suggested that the CD68 positive macrophage is one of the tumor-infiltrating macrophages in non-metastatic breast cancer. Following the anti-proliferative effect of ECCT exposure, the up-regulation of classical CD68 macrophage marker may involve the expression of chemokine related macrophage polarization. This argument is supported by Park et al.10, who reported that TTFs-induced inflammatory action is via the p38 MAPK/NF-kB signaling pathway and they believe that AC-EF based tumor therapy is a novel anticancer modality.\n\nSolid tumor interstitial fluid is an excellent environment for communication of signaling substances among stromal, macrophages and tumor cells. In this study (Figure 4), we evaluated the expression of CCL2, IL18, TNF-α, and IL23α, and whether they play a role in tumor progression or suppression. We demonstrated that the monocyte chemoattractant protein 1 (MCP-1) or CCL2 expression in solid breast tumor tissue with ECCT exposure (IT) is significantly lower (down-regulated) than without exposure (INT). There was no increase of relative mRNA CCL2 expression on ECCT exposure to the mammary gland of none DMBA induction rats (NIT) is shown in Figure 4I. This result supports a previous study that suggested that CCL2 has an essential role during breast cancer progression, through the induction of a systemic neutrophilic inflammatory cascade to facilitate metastasis37. Moreover, Lavender et al.38 reported that intra-tumoral CCL2 enables induction of breast tumor growth and/or metastases in breast cancer metastasis to the lung in vivo. Kirson et al.39 reported that TTFields (100 kHz and 1-2 Vpp) exposure has a potential action to inhibit the development of lung cancer metastasis from primary cancer cells. Additionally, IL18 and IL10 act synergistically on t angiogenesis progression8. Following CCL2 expression, the present result demonstrated that IL18 expression is downregulated in breast tumor tissue exposed to ECCT treatment (Figure 4C, D, J, and M). The high expression of IL18 in breast cancer enables promotion of cell proliferation and migration, and could be a biomarker candidate for prognosis in breast cancer patients40. Indeed IL-18 expression leads to invasion and metastasis of breast cancer cells through PI3K-AKT/ ATF-2 signaling, and it might be regulated through NF-κB/NF-κB1 signaling in TAMs41. Therefore, our result suggested that decreasing CCL2 and IL18 expression in the breast tumor microenvironment is most probably due to AC-EF treatment. We propose that there is a high correlation between the down-regulated expression of CCL2 and IL18 with the anti-proliferative effect of ECCT treatment. However, this argument should be further clarified by using other inflammatory markers of breast tumor progression.\n\nIn contrast with CCL2 and IL18 expression, in this study (Figure 4, M) the appearance of IL23α and TNF-α remains unchanged with the relative expression of both cytokines in normal mammary glands (NINT and NIT groups) and both solid breast tumor (INT and IT groups). A previous review42 reported that a higher TNF-α expression is related to breast tumor progression which is elevated in stage II, III and IV but not in stage I. IL23 is involved in inflammation and angiogenesis in response to cytotoxic T-cell infiltration in the tumor microenvironment. Therefore, in this study the relationship between IL23α and TNF-α expression, and anti-proliferative effect of AC-EF based tumor therapy remain unclear.\n\n\nConclusions\n\nIn conclusion, we purpose that noncontact ECCT treatment could have an anti-proliferative effect on solid breast tumor via the down-regulated expression of CCL2 and IL18. This preclinical study may become a basis of consideration for a clinical trial toward the implementation of ECCT as a novel cancer therapy modality.\n\n\nData availability\n\nOpen Science Framework: CCL2 and IL18 expressions may associate with the anti-proliferative effect of noncontact electro capacitive cancer therapy in vivo. https://doi.org/10.17605/OSF.IO/EP7KT43.\n\nThis project contains the following underlying data:\n\n- IHC data\n\n- Nodul data\n\n- Raw images for IHC and hematoxylin figures\n\n- Rat body weights during treatment\n\n- qPCR data\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nWe would like to thank M. Irfan, Finandya Fatihasari, Helmi Hana Prinanda, Ibnu Fajar, Miftah Jauhar, Puji Nurani, Windah Dwi Nuraini, Dalila Afinan, Ni Luh Kemmy Caesaria, Sevi Ratna Sari, Afif Yati and Pradana Putra Satya Negara for their kind helps during the works using animals, also many thanks to Mrs. Ardaning Nurliani for helping the antibodies procurement.\n\n\nReferences\n\nAlamsyah F, Ajrina IN, Dewi FNA, et al.: Antiproliferative Effect of Electric Fields on Breast Tumor Cells In Vitro and In Vivo. Indones J Cancer Chemoprevent. 2015; 6(3): 7. Publisher Full Text\n\nMujib SA, Alamsyah F, Taruno WP: Cell Death and Induced p53 Expression in Oral Cancer, HeLa, and Bone Marrow Mesenchyme Cells under the Exposure to Noncontact Electric Fields. Integr Med Int. 2017; 4(3–4): 161–70. Publisher Full Text\n\nKirson ED, Gurvich Z, Schneiderman R, et al.: Disruption of cancer cell replication by alternating electric fields. Cancer Res. 2004; 64(9): 3288–95. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPratiwi Rarastoeti: “CCL2 and IL18 Expressions May Associate with the Anti-proliferative Effect of Noncontact Electro Capacitive Cancer Therapy in Vivo.” OSF. 2019. http://www.doi.org/10.17605/OSF.IO/EP7KT" }	[ { "id": "55336", "date": "23 Oct 2019", "name": "Richard Luke Daniels", "expertise": [ "Reviewer Expertise Cell physiology", "Neuroscience", "Cell signaling" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 effects of intermediate frequency (100-300 kHz) alternative electric fields in a mouse model of breast cancer. Specifically, the authors examined expression (by immunohistochemistry and/or qPCR) of several genes. These were immune factors known to be associated with tumor cell proliferation and angiogenesis (CCL2 and IL18), a marker of cell proliferation (PCNA), a growth factor receptor (ErbB2), a marker of leukocyte differentiation (CD68) several other immune factors (IL18, IL23-alpha, TNF-alpha), and a marker of apoptosis (caspase-3). Changes were seen in gene expression consistent with decreased cell proliferation and increased cell death, though pro-inflammatory changes were not seen. These results provide evidence that supports a biological effect of alternating electric fields on cell proliferation, and it is this reviewer’s opinion that publication is warranted. There are a few minor modifications that I would suggest making before indexing this manuscript, which center around experiments described in figures 2-4.\n\nMinor English language corrections: In some instances, “none” should be changed to “non” throughout the document, as in “non DMBA-induced” (this is sometimes correctly stated, for example the animal methods section is correct). In the conclusion, change “purpose” to “propose”\nMethods: It would be beneficial to give more specifics about the protein quantification done using ImageJ. Was the scoring automated in anyway? What was the process or procedure used for determining whether a cell was positive for a given marker?\nFigure 2: The figure supports what is described in the text; however no quantification is given regarding the stated observations regarding the cell composition of the tumor and the structural changes with and without treatment. I do not consider this to be essential for indexing, however it would strengthen the conclusions drawn from this experiment if these results could be quantified.\n\nFigure 3: The figure supports what is described in the text. See comments related to the scoring method above. In the statistics, it is a bit unclear as to what was counted as an individual n. Was each slide counted as a single n? Was each image (50 images / slide)? It would be helpful for context to describe in a bit more detail either in these results or the methods section what was counted as an individual experiment for interpretation of the statistical comparisons.\nFigure 4: This figure supports what is described in the text. The graphs would benefit from changing the Y-axes to be consistently labeled with the same amount of significant figures. Also – if the CCL2 expression is 100-fold different than NIT, etc., it might be beneficial to point this out in the discussion, as this increase is substantially more than other differences seen that are 2-fold different, for example.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5728", "date": "23 Jul 2020", "name": "Rarastoeti Pratiwi", "role": "Author Response", "response": "External Reviewer (Dr. R. L. Daniels): This paper describes the effects of intermediate frequency (100-300 kHz) alternative electric fields in a mouse model of breast cancer. Specifically, the authors examined expression (by immunohistochemistry and/or qPCR) of several genes. These were immune factors known to be associated with tumor cell proliferation and angiogenesis (CCL2 and IL18), a marker of cell proliferation (PCNA), a growth factor receptor (ErbB2), a marker of leukocyte differentiation (CD68) several other immune factors (IL18, IL23-alpha, TNF-alpha), and a marker of apoptosis (caspase-3). Changes were seen in gene expression consistent with decreased cell proliferation and increased cell death, though pro-inflammatory changes were not seen. These results provide evidence that supports a biological effect of alternating electric fields on cell proliferation, and it is this reviewer’s opinion that publication is warranted. There are a few minor modifications that I would suggest making before indexing this manuscript, which center around experiments described in figures 2-4. Minor English language corrections: In some instances, “none” should be changed to “non” throughout the document, as in “non DMBA-induced” (this is sometimes correctly stated, for example the animal methods section is correct). In the conclusion, change “purpose” to “propose” User comment: All typo corrections have been done Methods: It would be beneficial to give more specifics about the protein quantification done using ImageJ. Was the scoring automated in anyway? What was the process or procedure used for determining whether a cell was positive for a given marker? User comment In the new version article has been revised: \"Data analysis for IHC was scored automatically using color deconvolution and computerized pixel profiling by IHC Profiler plugin on ImageJ version 1.51 software 29\" Figure 2: The figure supports what is described in the text; however no quantification is given regarding the stated observations regarding the cell composition of the tumor and the structural changes with and without treatment. I do not consider this to be essential for indexing, however it would strengthen the conclusions drawn from this experiment if these results could be quantified\". User comment: In the new version article has been revised:\"The tumor nodule is not a complex tissue, usually consist of connective and alveolar tissues, therefore the changing of cell composition is not clearly defined\". Figure 3: The figure supports what is described in the text. See comments related to the scoring method above. In the statistics, it is a bit unclear as to what was counted as an individual n. Was each slide counted as a single n? Was each image (50 images / slide)? It would be helpful for context to describe in a bit more detail either in these results or the methods section what was counted as an individual experiment for interpretation of the statistical comparisons. User comment In the new version article has been revised:\"The random 50 fields of view on IHC slides of each treatment were observed under Leica ICC50 E at 0.5 µm/pixel resolution\". Figure 4: This figure supports what is described in the text. The graphs would benefit from changing the Y-axes to be consistently labeled with the same amount of significant figures. Also – if the CCL2 expression is 100-fold different than NIT, etc., it might be beneficial to point this out in the discussion, as this increase is substantially more than other differences seen that are 2-fold different, for example. User Comment A revision has been done in the new version article:\"The appearance of CCL2(15.29 fold change), was significantly lower (p<0.01) on solid tumor tissues of the IT group than the INT group (97.72 fold change). This result was consistent with decreasing IL18 expression on tumor tissues of IT group (1.34 fold change) compared to the INT group (2.08 fold change)\". Figure 4.I. has been edited for the scale of expression relative (fold change). Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? No source data required Are the conclusions drawn adequately supported by the results? Partly Competing Interests No competing interests were disclosed. Reviewer Expertise Cell physiology, Neuroscience, Cell signaling" } ] }, { "id": "59978", "date": "05 Mar 2020", "name": "Yoram Palti", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Antara and colleagues reports on the use of noncontact ECCT treatment to down-regulated expression of CCL2 and IL18 and therefore inhibit breast cancer cell proliferation.\nComments:\nThe authors claim that the fluid bathing in breast tumors from ECCT-exposed IT treated rats may affect the tumors size, however, no specific analysis was done to support such claim. For instance, a single cell suspension of whole tumor followed by cell count could suffice. This data is crucial In order to support the claim that ECCT treatment is anti-proliferative, as tumor volume measurements show otherwise.\n\nThe authors should also elaborate on:\n\nThe impact of reduction in CCL2 and phenotype skewing in tumor macrophages. The discrepancy between CCL2 levels and CD68 infiltration. The discrepancy between past publications showing activation of the MAPK/NF-kB signaling pathway following AC-EF based tumor therapy (suggested as an example that has been used by the authors to support some of the conclusions in the current study) and the downregulation of IL18 in the current study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "65163", "date": "09 Jul 2020", "name": "Agung Putra", "expertise": [ "Reviewer Expertise Mesenchymal Stem Cell", "Immunology", "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 manuscript addresses the effects of noncontact electro capacitive cancer therapy (ECCT) to down-regulate CCL2 and IL18 expressions in a breast cancer mouse model in which the end targets is inhibiting breast cancer cell proliferation. It is an interesting topic in efforts to eliminate cancer cells by elaboration between the anti-proliferative effect and activity of inflammatory cells following ECCT administration to breast cancer, due to the conventional therapy to reduce recurrence and breast cancer mortality remains unclear. On the other hand, the proinflammatory cells releasing proinflammatory cytokines such as IL18, TNF-a, IL23, and chemokine CCL2 also play an essential role in the development of breast tumors.\n\nIn this paper, there are a number of issues regarding the methods and analysis that need to be clarified and addressed.\n\nBelow are more specific comments by section:\n\nIntroduction:\nMinor corrections:\nadd a punctuation mark \"comma\" in this sentence:\nStromal components, such as endothelial cells, myeloid derivate suppressor cells, and macrophages”\nadd “the” in this sentences:\nFurthermore, the evaluation of IL-23 suggested that this cytokine has… “\nAdd “-“ in this sentences:\nand growth by up-regulating matrix metalloproteinase….”\nAdd “a” in this sentences\nMCSs into a wound site and allow macrophages to be at a short distance to the wound…”\n\n“Macrophages and tumor cells act as inflammatory cells that can be affected by chemical signals.” Tumor cells is not inflammatory cells.\n\nSo far, electric fields-based therapies need to be described in order to understand the modulation. The mechanism need to be investigated.\n\n“TNF-α significantly induces breast cancer metastasis via TNFα-activated mesenchyme stem cells (MSCs) in lung.” However, under TNF-α stimulation at invitro model, the MSCs can also release IL-10 as anti-inflammatory cytokine that may be a beneficial for cancer growth inhibition.\nPlease read this article: Putra A, Ridwan FB, Putridewi AI, et al. The Role of TNF-α induced MSCs on Suppressive Inflammation by Increasing TGF-β and IL-10. Open Access Maced J Med Sci. 2018;6(10):1779-1783. Published 2018 Oct 4. doi:10.3889/oamjms.2018.404[ref-1].\n“Tumor-associated macrophages (TAMs) may help tumor cell growth by releasing several pro-inflammatory cytokines, such as TNFα and IL23, which are expressed by the classically activated M1 macrophage.” Whereas, TAM is not classically activated M1 macrophage but the TAM is definitely an activated M2 macrophage.\nRead this article: Lee C, Jeong H, Bae Y, et al. Targeting of M2-like tumor-associated macrophages with a melittin-based pro-apoptotic peptide. J Immunother Cancer. 2019;7(1):147. Published 2019 Jun 7. doi:10.1186/s40425-019-0610-4[ref-2].\nA recent study demonstrated that TTFields therapy activates macrophage specific immune responses through the activation of several cytokines, such as NF-kB (nuclear factor-kB), TNF-α, and IL1-β….. NF-kB is not cytokine but the NF-Kb is the rapid-acting primary transcription factor for controlling cytokine production.\n\nMethod:\nMinor corrections:\nAdd “this” in the sentence:\nThe rat number for this experimental design was calculated for the minimal biological replication of rats…. “\nAdd “a” in the sentence:\nTumor nodule diameters were measured every 2 days using a digital caliper ….”\n\nThe sample paraffin blocks were sectioned with a microtome...”\n\nThe stained samples were subsequently dehydrated using an upgraded level of...”\nAdd “-“ in the sentence: Solid tumor-bearing rats were exposed…. Add “comma” in the sentence:\nusing an upgraded level of alcohol, cleared in xylene, and lastly”\nRemove “s” in the sentence:\nThe INT and IT tumor tissues samples were then processed…”\nAdd “a” and “-“ in the sentence:\nIn brief, the samples were deparaffinized using xylene and then rehydrated with a down-graded concentration of alcohol….”\n\nPlease clarify the experimental group. The experimental design of this study used four rat groups with six biological replicates (n=6), however, the number of tissue sample required for replication is three tissues samples (n=3)...”\n\nResults\nThe authors reported that ECCT-exposed rat breast tumor (IT) can induce the fluid bathing in tumors that associated with tumors size, in which the author's hypothesis is to inhibit breast tumor cell proliferation (anti-proliferation). Although, the results from the sham rat breast tumor group (INT) exhibited denser tissues and slower growth, however, the tumor volume of IT (treatment group) still exhibited larger than the sham (INT group), suggesting the result is on the contrary to the anti-proliferation. (Figure 1). Please clarify.\n\nThe authors declared that the percentage of cells expressing CD68 protein in treatment groups (IT) were significantly higher than in the non-treatment groups (INT group) indicated that the macrophage polarized into type-2, known as Tumor associated macrophage (TAM) that associated with the proliferation of cancer cell. Please clarify.\n\nOn the other hand, in this study there was a decrease of CCL2 as the main chemokine of monocyte and macrophages, however the macrophage type-2 (CD 68 expression) was increased, Please clarify.\n\nIs the work 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": "5729", "date": "23 Jul 2020", "name": "Rarastoeti Pratiwi", "role": "Author Response", "response": "External Reviewer (Dr. A. Putra): APPROVED WITH RESERVATIONS This manuscript addresses the effects of noncontact electro capacitive cancer therapy (ECCT) to down-regulate CCL2 and IL18 expressions in a breast cancer mouse model in which the end targets is inhibiting breast cancer cell proliferation. It is an interesting topic in efforts to eliminate cancer cells by elaboration between the anti-proliferative effect and activity of inflammatory cells following ECCT administration to breast cancer, due to the conventional therapy to reduce recurrence and breast cancer mortality remains unclear. On the other hand, the proinflammatory cells releasing proinflammatory cytokines such as IL18, TNF-a, IL23, and chemokine CCL2 also play an essential role in the development of breast tumors. In this paper, there are a number of issues regarding the methods and analysis that need to be clarified and addressed. Below are more specific comments by section: Introduction: Minor corrections: add a punctuation mark \"comma\" in this sentence: Stromal components, such as endothelial cells, myeloid derivate suppressor cells, and macrophages” add “the” in this sentences: Furthermore, the evaluation of IL-23 suggested that this cytokine has… “ Furthermore, evaluation of IL-23 suggested Add “-“ in this sentences: and growth by up-regulating matrix metalloproteinase….” and growth by up regulating Add “a” in this sentences MCSs into a wound site and allow macrophages to be at a short distance to the wound…” MCSs into a wound site and allow macrophages to be at short distance to the wound. “Macrophages and tumor cells act as inflammatory cells that can be affected by chemical signals.” Tumor cells is not inflammatory cells. User comment: All typo corrections have been done. So far, electric fields-based therapies need to be described in order to understand the modulation. The mechanism need to be investigated. User comment: Revision has been done. In the new version article has been added “The mechanism needs to be investigated”. “TNF-α significantly induces breast cancer metastasis via TNFα-activated mesenchyme stem cells (MSCs) in lung.” However, under TNF-α stimulation at invitro model, the MSCs can also release IL-10 as anti-inflammatory cytokine that may be a beneficial for cancer growth inhibition. Please read this article: Putra A, Ridwan FB, Putridewi AI, et al. The Role of TNF-α induced MSCs on Suppressive Inflammation by Increasing TGF-β and IL-10. Open Access Maced J Med Sci. 2018;6(10):1779-1783. Published 2018 Oct 4. doi:10.3889/oamjms.2018.404[ref-1]. User comment: In the new version, a revision has been done by added a new reference: reference no 16. “Tumor-associated macrophages (TAMs) may help tumor cell growth by releasing several pro-inflammatory cytokines, such as TNFα and IL23, which are expressed by the classically activated M1 macrophage.” Whereas, TAM is not classically activated M1 macrophage but the TAM is definitely an activated M2 macrophage. Read this article: Lee C, Jeong H, Bae Y, et al. Targeting of M2-like tumor-associated macrophages with a melittin-based pro-apoptotic peptide. J Immunother Cancer. 2019;7(1):147. Published 2019 Jun 7. doi:10.1186/s40425-019-0610-4[ref-2]. User comment: In the new version, a revision has been done by added a new reference: reference no 19. 3. A recent study demonstrated that TTFields therapy activates macrophage specific immune responses through the activation of several cytokines, such as NF-kB (nuclear factor-kB), TNF-α, and IL1-β….. NF-kB is not cytokine but the NF-Kb is the rapid-acting primary transcription factor for controlling cytokine production. User comment: Revision has been done by deleting: “NF-kB (nuclear factor-kB),” Method: Minor corrections: Add “this” in the sentence: The rat number for this experimental design was calculated for the minimal biological replication of rats…. “ Add “a” in the sentence: Tumor nodule diameters were measured every 2 days using a digital caliper ….” The sample paraffin blocks were sectioned with a microtome...” The stained samples were subsequently dehydrated using an upgraded level of...” The stained samples were subsequently dehydrated using upgraded level Add “-“ in the sentence: Solid tumor-bearing rats were exposed…. Add “comma” in the sentence: using an upgraded level of alcohol, cleared in xylene, and lastly” using an upgraded level of alcohol, cleared in xylene and, lastly, Remove “s” in the sentence: The INT and IT tumor tissues samples were then processed…” Add “a” and “-“ in the sentence: In brief, the samples were deparaffinized using xylene and then rehydrated with a down-graded concentration of alcohol….” User comment: In the new version, all these typo corrections have been done. Please clarify the experimental group. The experimental design of this study used four rat groups with six biological replicates (n=6), however, the number of tissue sample required for replication is three tissues samples (n=3)...” User comment: Revision: In the new version, the (n=3) have been deleted in the explanations of:” · - NINT group: non-DMBA-induced rats, non-therapy exposure (n=3) · - NIT group: non-DMBA-induced rats, therapy exposure (n=3) · - INT: DMBA-induced rats, non-therapy treatment (n=3) · - IT: DMBA-induced rate, therapy exposure (n=3)”, because it could confuse. In the new version, the replication of rat (n=6) and three tissues sample per treatment group (n=3) have been added in the text. So, the NINT, NIT, INT and IT just to explain the treatment for each group (without (n=3). Clarification: As I described in the manuscript: “The rat number for this experimental design was calculated for the minimal biological replication of rats, with four treatment groups according to the Federer Formula 23 “ and this number also follow the ethical procedure for using the minimal animals. However, we only using a minimal number of tissue samples replication requirement for statistical analysis in random sampling method. Three tissues samples per treatment group (4 groups) was used for a biological replication requirement. Each sample was triple sections per tissue sample for histopathologic scoring requirements (to gain 50 fields of view) and twice for qRT-PCR analysis. However, we used six rats (n=6) per group to observe rat body weights (secondary data) Results 1. The authors reported that ECCT-exposed rat breast tumor (IT) can induce the fluid bathing in tumors that associated with tumors size, in which the author's hypothesis is to inhibit breast tumor cell proliferation (anti-proliferation). Although, the results from the sham rat breast tumor group (INT) exhibited denser tissues and slower growth, however, the tumor volume of IT (treatment group) still exhibited larger than the sham (INT group), suggesting the result is on the contrary to the anti-proliferation. (Figure 1). Please clarify. User comment: Clarification: The larger size of tumor nodules in IT groups due to the measurement using a digital caliper. In that case, we measured the nodule volume included the inflammation fluids, instead of tumor tissues. However, from the histological observation, the results showed that the number of proliferation cells in IT groups significantly lower than in INT group. So, from this result we suggest that the tumor interstitial fluid contains stromal and immune cells could increase the tumor nodule volume. Further, we observed the proliferation of the tumor cells (based on PCNA marker of cells), and the role of macrophage (based on CD68 marker of monocyte and macrophage) to eliminate the tumor cells which are failure to divided. 2. The authors declared that the percentage of cells expressing CD68 protein in treatment groups (IT) were significantly higher than in the non-treatment groups (INT group) indicated that the macrophage polarized into type-2, known as Tumor associated macrophage (TAM) that associated with the proliferation of cancer cell. Please clarify. User comment: Revision: In the new revision, the words “a classical”, in the sentence: ” ... expression of CD68 (a classical macrophage marker)..” has been deleted and edited: “…expression of CD68 (a marker for macrophage and monocyte) Clarification: CD68 is the marker of macrophage and monocyte (see the revision obove), so these macrophages could be M1 and M2 (TAM). 3. On the other hand, in this study there was a decrease of CCL2 as the main chemokine of monocyte and macrophages, however the macrophage type-2 (CD 68 expression) was increased, Please clarify. User comment: Clarificasion: CD68 can be expressed either in monocyte and macrophage (M1 and M2). However, CCL2 level correlated with TAM abundance and TAM is M2 Macrophage. The results show that the increase of macrophage CD68 (could be M1 and M2 macrophages) in IT group, but the expression of CCL2 in this group is decrease. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests No competing interests were disclosed. Reviewer Expertise Mesenchymal Stem Cell, Immunology, Cancer I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above." } ] } ]	1	https://f1000research.com/articles/8-1770
https://f1000research.com/articles/9-334/v1	06 May 20	{ "type": "Data Note", "title": "A dataset for the perceived vulnerability to disease scale in Japan before the spread of COVID-19", "authors": [ "Yuki Yamada", "Haoqin Xu", "Kyoshiro Sasaki", "Haoqin Xu", "Kyoshiro Sasaki" ], "abstract": "The COVID-19 outbreak is a worldwide medical and epidemiological catastrophe, and the number of psychological studies concerning COVID-19 is growing daily. Such studies need baseline data from before the COVID-19 outbreak for comparison, but such datasets have not yet been accumulated and shared. Here, we provide a dataset on the perceived vulnerability to disease scale for 1382 Japanese participants obtained through an online survey conducted in 2018 that will be useful for comparison with current or post-COVID-19 perceived vulnerability to disease data.", "keywords": [ "coronavirus", "disgust", "emotion", "Japanese", "perceived infectability", "germ aversion" ], "content": "Introduction\n\nCurrently, a new type of coronavirus infection (COVID-19) is spreading on a global scale. Although the details of this infection are not yet clear, it is rapidly spreading in many countries, and the strength of the infection is likely to be great. In response to this unprecedented situation, governments are asking people to take social distancing measures and limit their outside activities. Under such threats and political measures, the state of mind of the people must also be quite different from before. Therefore, many social and behavioral studies of such factors including people’s political attitudes, controllability, emotional states, anxiety, and stress in COVID-19 situations are being conducted simultaneously and rapidly (Van Bavel et al., 2020). However, it is difficult to predict how long the current pandemic will last, and even if the situation is under control, it is unclear whether the psychological traits of people in the post-COVID-19 world who experienced this pandemic will be similar to those before COVID-19. Thus, such survey studies need pre-outbreak data as a baseline for comparison and data obtained before the COVID-19 outbreak are of great importance. Accordingly, we here provide a dataset for the perceived vulnerability to disease scale obtained in Japan in 2018 (Yamada, 2020).\n\nWith the spread of COVID-19 and under the guidance of the World Health Organization (WHO), people have begun to wash their hands more often. Concomitantly, we have become more afraid of infection than ever before. People have begun to disinfect various places and objects with alcohol and to wear masks. These behaviors are based on a heightened perceived vulnerability to disease (PVD). A psychological scale has been developed to measure this tendency (Duncan et al., 2009). The PVD scale is composed of two subscales: “perceived infectability,” which is related to the beliefs of one’s own susceptibility to infecting diseases, and “germ aversion,” which is related to an awareness of discomfort in situations with a high likelihood of infection with a pathogen. This scale has already been localized in Japan (Fukukawa et al., 2014). It has also been translated not only in Japan but other countries as well (Ahmadzadeh et al., 2013; Klavina et al., 2011; Prokop et al., 2010; Skolnick & Dzokoto, 2013). In the early stages of the COVID-19 spread (i.e., January 2020), a Chinese study had already compared PVD scale scores with those of other countries (Goh, 2020). These results suggest that the generality of this scale and the necessity of baseline data are both striking. Therefore, we provide data on the pre-pandemic PVD scale for use in comparative studies (Yamada, 2020).\n\n\nMethods\n\nWe recruited a maximum of 2000 participants through Yahoo! Crowdsourcing Service and recorded the data collected during the survey period. As a result, a total of 1428 Japanese people in Japan participated in this survey (868 men, 543 women, 17 unknown; mean age 43.40 years).\n\nWe used the Japanese version of the PVD scale developed by Fukukawa et al. (2014). The scale consists of a total of 15 items. Each item was scored on a seven-point scale (1: strongly disagree, 7: strongly agree). Items 3, 5, 11, 12, 13, and 14 were reverse-scaled items. All the items of this scale are available from the original papers (English: Duncan et al., 2009; Japanese: Fukukawa et al., 2014).\n\nThe survey was conducted from September 22–23, 2018. Participants accessed the Yahoo! Crowdsourcing service page for the link to the web address of the survey page on Google Forms. The participants were first asked to input their age and sex (male, female, or other). The order of items on the scale was randomized across participants based on the setting of Google Forms. In order to check whether the participants were concentrating on the task, a calculation problem (171 − 169 = ?) was inserted as an attention check question to identify respondents who do not answer seriously (Sasaki & Yamada, 2019). After the survey, the participants received 10 T-points (Japanese point service, in which one T-point is worth one JPY) as a reward.\n\nThe survey was posted on the website of the crowdsourcing service and users of the service were free to view it and participate in it.\n\nWe excluded participants who gave an incorrect answer to the attention check question. As a result, we eliminated the data of 46 participants. We present the remaining dataset for 1382 participants (833 men, 533 women, 16 unknown; mean age 43.46 years) as a relatively reliable one.\n\nThe present study received approval from the psychological research ethics committee of the Faculty of Human-Environment Studies at Kyushu University (approval number: 2016-017). Completion of the survey was taken as consent to participate from participants. Participants had the right to withdraw from the survey at any time without providing a reason. Although we did not obtain personal information about the participants, as this was a crowdsourced survey, it was explained to them that their responses would not be tied to them personally.\n\n\nData availability\n\nOpen Science Framework: Japan PVD 2018. https://doi.org/10.17605/OSF.IO/QW2AF; registration DOI https://doi.org/10.17605/OSF.IO/7Y4AV (Yamada, 2020).\n\nThis project contains the following underlying data:\n\nPVDJapan2018.xlsx. (The dataset.)\n\nDescription of Dataset.txt.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nWe would like to thank Editage (www.editage.com) for English language editing.\n\n\nReferences\n\nAhmadzadeh M, Ghamarani A, Samadi M, et al.: The investigation of validity and reliability of a scale of perceived vulnerability to disease in Iran. British Journal of Social Sciences. 2013; 1(4): 43–51. Publisher Full Text\n\nDuncan LA, Schaller M, Park JH: Perceived vulnerability to disease: Development and validation of a 15-item self-report instrument. Personality and Individual Differences. 2009; 47(6): 541–546. Publisher Full Text\n\nFukukawa Y, Oda R, Usami H, et al.: [Development of a Japanese version of the Perceived Vulnerability to Disease Scale]. Shinrigaku Kenkyu. 2014; 85(2): 188–195. PubMed Abstract \| Publisher Full Text\n\nGoh JX: Perceived vulnerability to disease predicts restrictive policy supports in response to the 2019-nCoV outbreak. 2020. Publisher Full Text\n\nKlavina L, Buunk AP, Pollet TV: Out-group mating threat and disease threat increase implicit negative attitudes toward the out-group among men. Front Psychol. 2011; 2: 76. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nProkop P, Usak M, Fančovičová J: Risk of parasite transmission influences perceived vulnerability to disease and perceived danger of disease-relevant animals. Behav Processes. 2010; 85(1): 52–57. PubMed Abstract \| Publisher Full Text\n\nSasaki K, Yamada Y: Crowdsourcing visual perception experiments: a case of contrast threshold. PeerJ. 2019; 7: e8339. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSkolnick AJ, Dzokoto VA: Disgust and contamination: a cross-national comparison of ghana and the United States. Front Psychol. 2013; 4: 91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVan Bavel JJ, Baicker K, Boggio PS, et al.: Using social and behavioural science to support COVID-19 pandemic response. Nat Hum Behav. 2020. PubMed Abstract \| Publisher Full Text\n\nYamada Y: Japan PVD 2018. 2020. http://www.doi.org/10.17605/OSF.IO/7Y4AV" }	[ { "id": "63128", "date": "26 May 2020", "name": "Shu Imaizumi", "expertise": [ "Reviewer Expertise Cognitive psychology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors provide a dataset of the PVD scale from a Japanese sample in September 2018, which would serve as a baseline for comparison with the PVD in the current and post COVID-19 situations. The study will give not only a dataset but also an important message for future studies on the changes in human behavior during and after this COVID-19 period. I would like to give some minor comments.\n\nIntroduction:\nTo be precise, COVID-19 is an abbreviation for coronavirus disease 2019.\n\n“perceived infectability” and “germ aversion” may be capitalized, according to Duncan et al. (2009).\n\nIt might be better to clarify that Goh (2020) was a cross-sectional study (i.e., no comparison with pre-COVID-19 data) to underscore the necessity of baseline.\nParticipants, Exclusion:\nIt would be better to report SD of participants’ age.\n\nProcedure:\nPlease clarify whether the calculation problem was inserted between or after the PVD scale items.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "5730", "date": "22 Jul 2020", "name": "Yuki Yamada", "role": "Author Response", "response": "The authors provide a dataset of the PVD scale from a Japanese sample in September 2018, which would serve as a baseline for comparison with the PVD in the current and post COVID-19 situations. The study will give not only a dataset but also an important message for future studies on the changes in human behavior during and after this COVID-19 period. I would like to give some minor comments. Reply: Thank you very much for your positive and valuable comments. We want to address your remarks with sincerity. To be precise, COVID-19 is an abbreviation for coronavirus disease 2019. “perceived infectability” and “germ aversion” may be capitalized, according to Duncan et al. (2009). Reply: Following your suggestions, we have revised the manuscript accordingly. It might be better to clarify that Goh (2020) was a cross-sectional study (i.e., no comparison with pre-COVID-19 data) to underscore the necessity of baseline. Reply: Thank you for this valuable suggestion. We have clarified that Goh (2020) was a cross-sectional study in the revised manuscript. It would be better to report SD of participants’ age. Reply: We have calculated the SD (= 10.62) of participants’ age and indicated this in the revised manuscript. Please clarify whether the calculation problem was inserted between or after the PVD scale items. Reply: Based on this comment, we have clarified that the calculation problem was inserted after the 7th item of the PVD scale." } ] }, { "id": "64012", "date": "14 Jul 2020", "name": "Amir H. Pakpour", "expertise": [ "Reviewer Expertise Psychology", "methodology", "Biostatistics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDear Author(s), Please\n\nThe aim of this study is vague. The abstract is incomplete and the results and the method parts are not accessible. In addition to this, your keywords are not able to reflect the whole details of the study.\n\nOne of the psychological scales was introduced, however, there is not any hint to find out about that.\n\nIn the method part, there is not solid ground to opt Web-sampling? it could lead to bias definitely.\nIs there any previous knowledge to select age and sex as covariates?\n\nI would suggest the authors add the following citation: Ahorsu, D. K., Lin, C. Y., Imani, V., Saffari, M., Griffiths, M. D., & Pakpour, A. H. (2020). The fear of COVID-19 scale: development and initial validation. International journal of mental health and addiction.1\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and materials provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "5731", "date": "22 Jul 2020", "name": "Yuki Yamada", "role": "Author Response", "response": "Thank you for reviewing our data paper. We were happy to address each comment. 1. The aim of this study is vague. The abstract is incomplete and the results and the method parts are not accessible. In addition to this, your keywords are not able to reflect the whole details of the study. Reply: Thank you for your comment. Our manuscript was submitted as Data Notes, in which, the paper aims to provide a valuable dataset and promote its reuse. Further, this point was already apparent in the first draft. Generally, Data Notes should explain how the data was created without including any analyses or conclusions. As well, we followed the article guidelines, which are available at https://f1000research.com/for-authors/article-guidelines. Therefore, based on the guidelines, we believe that our abstract was not incomplete. 2. One of the psychological scales was introduced, however, there is not any hint to find out about that. Reply: The present study used only the Japanese version of the perceived vulnerability to disease (PVD) scale (Fukukawa et al., 2014); we assume that you are referring to this scale. This scale is a just localized version of the original PVD scale (Duncan et al., 2009); the contents in the Japanese version of the PVD scale are also identical to the original scale. Furthermore, we already clarified this point in the Introduction of the first draft. 3. n the method part, there is not solid ground to opt Web-sampling? it could lead to bias definitely. Reply: Thank you very much for your remark. Although it is unclear to us what kind of bias you were referring to regarding the web sampling, we believe that those who will use this dataset will carefully address any areas of concern. To that end, we explicitly stated that this data was obtained by web-sampling and provided more detailed data characteristics, as responded in 2-4. 4. Is there any previous knowledge to select age and sex as covariates? Reply: As per our response in 2-1, Data Notes do not have to include any analyses or conclusions. Moreover, age and sex as covariates are dependent on the purpose of using this dataset and should not be predicated here; therefore, those using the dataset should address age and sex as covariates as they deem necessary. But just for information, we would show a graph representing the relation between the individual PVD scale score and age for each gender for future data users. 5. I would suggest the authors add the following citation: Ahorsu, D. K., Lin, C. Y., Imani, V., Saffari, M., Griffiths, M. D., & Pakpour, A. H. (2020). The fear of COVID-19 scale: development and initial validation. International journal of mental health and addiction.1 Reply: Thank you for this valuable information. We have cited this article in the revised manuscript. We express our sincere gratitude for your time and effort for the valuable comments and suggestions, and hope that our revised manuscript is now suitable for approval." } ] } ]	1	https://f1000research.com/articles/9-334
https://f1000research.com/articles/9-761/v1	22 Jul 20	{ "type": "Systematic Review", "title": "Diagnostic test accuracy of Xpert MTB/RIF for tuberculous pericarditis: a systematic review and meta-analysis", "authors": [ "Andrianto Andrianto", "Ni Made Mertaniasih", "Parama Gandi", "Makhyan Jibril Al-Farabi", "Yusuf Azmi", "Michael Jonatan", "Stevanus Immanuel Silahooij", "Makhyan Jibril Al-Farabi", "Yusuf Azmi", "Michael Jonatan" ], "abstract": "Introduction: Xpert MTB/RIF is a rapid diagnostic instrument for pulmonary tuberculosis (TB). However, studies reported varied accuracy of Xpert MTB/RIF in detecting Mycobacterium tuberculosis in pericardial effusion. Methods: We performed a systematic review of literature in PubMed, published up to February 1, 2020, according to PRISMA guidelines. We screened cross-sectional studies, observational cohort studies, and randomized control trials that evaluated the accuracy of Xpert MTB/RIF in diagnosing TB pericarditis. Papers with noninterpretable results of sensitivity and specificity, non-English articles, and unpublished studies were excluded. The primary outcomes were the sensitivity and specificity of Xpert MTB/RIF. We conducted a quality assessment using QUADAS-2 to evaluate the quality of the studies. A bivariate model pooled the overall sensitivity, specificity, positive likelihood ratios (PLRs), and negative likelihood ratios (NLRs) of included studies. Results: In total, 581 subjects from nine studies were analyzed in this meta-analysis. Our pooled analysis showed that the overall sensitivity, specificity, PLRs and NLRs of included studies were 0.676 (95% CI: 0.580–0.759), 0.994 (95% CI: 0.919–1.000), 110.11 (95% CI: 7.65–1584.57) and 0.326 (95% CI: 0.246–0.433), respectively. Conclusions: Xpert MTB/RIF had a robust specificity but unsatisfactory sensitivity in diagnosing TB pericarditis. These findings indicated that although positive Xpert MTB/RIF test results might be valuable in swiftly distinguishing the diagnosis of TB pericarditis, negative test results might not be able to rule out TB pericarditis. Registration: PROSPERO CRD42020167480 28/04/2020", "keywords": [ "Extrapulmonary TBC", "Pericardial Effusion", "PCR", "Xpert MTB/RIF" ], "content": "Introduction\n\nPericarditis tuberculosis (TB) is the deadliest manifestation of extrapulmonary TB. TB is the primary cause of clinically signiﬁcant pericardial effusion in TB-endemic developing countries, responsible for as much as 90% of pericardial effusion in HIV-infected individuals and 50–70% in non-HIV-infected individuals1. Mortality rates due to pericarditis TB rise six months after diagnosis, from 17% to 40%2.\n\nEarly and accurate diagnostic measures are essential for tackling deadly extrapulmonary TB infections3. Until 2018, Lowenstein-Jensen culture (LJ) was considered the gold standard for diagnosing TB pericarditis, since early non-invasive diagnostic procedures such as chest radiographs can only expose changes indicative of TB in 30% of cases, and echocardiography only presents a large frond-like pericardial effusion projection, which is nowhere near specific enough to indicate TB etiology4,5. However, LJ culture is time-consuming and complicated. Thus it is not recommended for use as a routine test6. Other alternatives, such as histopathological examination, require invasive procedures and expertise that can only be found in a few medical centers. In addition, its sensitivity remains highly variable, ranging from 30 – 70%7. Thus, histopathological examination is also not recommended for use as a routine test8.\n\nCurrently, the Xpert MTB/RIF assay has FDA-approval as a diagnostic test of pulmonary TB. The Xpert MTB/RIF assay can detect members of the Mycobacterium complex and rifampicin resistance via nucleic acid amplification. This assay has been proven useful to diagnose pulmonary TB, with 89% sensitivity and 99% specificity7. However, this method has not been established as a diagnostic test for extrapulmonary TB, such as pericarditis7.\n\nSeveral studies and meta-analyses have explored the benefits of using the Xpert MTB/RIF assay to diagnose extrapulmonary TB. However, to our knowledge, there is no meta-analysis of the use of the Xpert MTB/RIF assay in diagnosing pericarditis TB. Varied results, ranging from 59%–100% sensitivity and 72–100% specificity, have been found for the use of the Xpert MTB/RIF assay to diagnose pericarditis. Thus, this study intended to evaluate the accuracy of TB pericarditis diagnosis using a pericardial fluid sample for the Xpert MTB/RIF assay.\n\n\nMethods\n\nA meta-analysis was performed from February to April 2020 to assess the specificity and sensitivity of Xpert MTB/RIF for the diagnosis of pericarditis TB. This research was conducted according to the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) statement and Cochrane Handbook for Systematic Reviews of Interventions. A checklist and review protocol adapted from PRISMA was used to guide the meta-analysis protocols in our present study (see Extended data). The systematic review protocol was registered with the International Prospective Register of Systematic Reviews (PROSPERO; identiﬁcation number CRD42020167480) on 28/04/2020.\n\nAny document reporting diagnosis of pericarditis TBC using Xpert MTB/RIF assay based on primary data was considered. We included cross-sectional studies, observational cohort studies, and randomized control trials that evaluated the diagnostic accuracy of Xpert MTB/RIF for TB pericarditis. Exclusion criteria were (i) non-English articles; (ii) review, case-control studies, and case reports; (iii) studies that did not contain primary data in the published articles; and (iv) studies with noninterpretable results of sensitivity and specificity or had no samples that showed true positive (TP) and false positive (FP) results and/or true negative (TN) and false negative (FN) results. Duplicate data, commentaries, letters, correspondence articles, and editorials were excluded. Unpublished records such as conference papers, theses, and patents were not included in this meta-analysis.\n\nArticles were systematically searched from inception to February 1, 2020, in PubMed databases. The search strategy conformed to medical subjects heading (MeSH) involving the use of the following keywords: (\"tuberculous AND pericarditis\") OR (\"extrapulmonary OR pericardial OR pericardium\") AND (tuberculosis) AND (\"Xpert OR genexpert\"). In our searching strategy, English language restrictions were applied. If the same data were used among studies, the more up-to date study with the larger sample size was preferred. The references cited by all the selected original research articles and reviews were searched for additional articles that might have been missed. Authors independently reviewed abstracts (Y.A, M.J, P.G, M.J.A, S.I.S) obtained from database searches and selected potentially eligible studies for full-text reviews. Data from included studies were independently extracted from eligible articles using a piloted data extraction form. The review authors extracted data independently on the following characteristics: name of the first author, year of publication, country of subjects, type of data collection, sampling method, patient characteristics and setting, the procedure for processing specimens, reference standards, and the results of the calculation of diagnostic test accuracy. All data were entered into a database manager, Microsoft Excel 2014. Disagreement between independent investigators was resolved by discussion and/or by consultation with the senior investigators (A, N.M.M).\n\nTo ensure quality and to avoid potential bias in each study, the quality of selected studies was controlled and assessed by two independent investigators (M.J.A, S.I.S). Included studies were appraised for their quality and bias using QUADAS-2. QUADAS-2 comprises four independent areas: subject preference, index test, source standard, and the flow and timing of patients through the study.\n\nWe used the TP, FN, FP, and TN values reported in each included study to determine sensitivity and specificity. We used 95% confidence intervals (CIs) and generated the results graphically by plotting the data in forest plots. We plotted continuous diagnostic test results versus a gold standard in summary receiver operating characteristic (sROC) curves using Review Manager 5 (RevMan Cochrane, London, UK). The overall diagnostic sensitivity and specificity of included studies were pooled by a bivariate model using STATA 12 (STATA Corp, USA) with the \"metandi\" command.\n\n\nResults\n\nAfter conducting literature screening, we included 30 studies for full-text article assessment. However, one study had different reference diagnoses, two studies were not in English, ten studies did not contain primary data in their published article, and eight studies had inadequate samples to calculate sensitivity and specificity; thus, they were excluded from our study. Therefore, in this meta-analysis, we included nine studies9–17. Figure 1 showed the flowchart of the research collection, while Table 1 provided a summary of the included studies. Of the nine studies included, three were carried out in Pakistan10,13,15, two were conducted in South Africa12,14, and the rest were carried out in Turkey17, Iran9, China16, and Spain11. All studies used a prospective observational design. The number of pericardial effusion samples from each study ranged from three to 131, with a total sample size of 581 patients. All studies used a sample of patients with pericardial effusion, with an average prevalence of TB pericarditis of 28.4%. The MTB/RIF assay was applied to test the sensitivity and specificity in detecting Mycobacterium tuberculosis and resistance to rifampicin. The MTB/RIF assay ran nucleic acid amplification of the rpoB gene to detect M. tuberculosis, while resistance to rifampicin was assessed in an 81 bp region within the rpoB gene. As a diagnostic reference for MTB/RIF, a culture of pericardial fluid was used with solid and liquid media.\n\nEPTB, extrapulmonary tuberculosis; PTB, pulmonary tuberculosis; PeE, pericardial effusion; PuE, pleural effusion TB, tuberculosis; NR, not reported.\n\nWe conducted a quality appraisal of included studies using QUADAS-2, and the results were shown in Figure 2 and Figure 3. QUADAS-2 examined four areas of the study, namely subject preference, index tests, source standards, and flow and timing. Each area, except flow and timing, consisted of two subdivisions, namely, risk of bias and applicability concerns related to patient characteristics and settings.\n\nRegarding the risk of bias in the subject preference area, six studies9,10,12,14,16,17 were labeled as low risk, two studies13,15 were labeled as high risk, and one study11 was labeled as an unclear risk because it did not explain the exclusion criteria clearly. The reason for the high risk of bias in this domain was due to inappropriate exclusion criteria. In the applicability concern section, we labeled one study as a low concern12, three studies10,13,14 as high concerns, and five studies9,11,15–17 as unclear concerns since the setting of patients was not mentioned. Studies in primary health settings were deemed low concern, whereas studies set exclusively in a tertiary care center were deemed high concern.\n\nIn the index test area, all studies included9–17 were labeled as having a low risk of bias. This was because Xpert MTB/RIF provided automatic results with a prespecified threshold test, and the user received the results on a printed sheet. In the applicability concern section, we grouped seven studies as low concerns9–12,14 and two studies as high concerns15,17. To be categorized as a low concern, a minimum of 75% of the specimens in the study were required to have been processed according to WHO recommendations.\n\nIn the reference standard area, eight studies9–12,14–17 were labeled as having a low risk of bias, and one study13 had an unclear risk of bias. To be categorized as a low risk of bias, the results of reference standards must be determined without the knowledge of the outcomes of index tests.\n\nIn the flow and timing area, all studies9–17 were considered to have a low risk of bias because all studies included more than 50% of eligible participants in the analysis.\n\nTable 2 showed the significant findings from the included studies. In general, Xpert MTB/RIF had a high diagnostic accuracy in diagnosing TB pericarditis. The diagnostic test sensitivity and specificity forest plot of Xpert MTB/RIF for TB pericarditis were shown in Figure 4. Xpert MTB/RIF had an overall sensitivity of 0.676 (95% CI: 0.580–0.759), and the overall specificity was 0.994 (95% CI: 0.919–1.000). The PLR and NLR were 110.11 (95% CI: 7.65–1584.57) and 0.326 (95% CI: 0.246–0.433), respectively. Figure 5 showed a summary receiver operating characteristic (sROC) for Xpert MTB/RIF to illustrate continuous diagnostic test results versus the gold standard.\n\nTP, true positive; FP, false positive; FN, false negative; TN true negative.\n\nTP, true positive; FP, false positive; FN, false negative; TN, true negative.\n\nThe black curve describes a pattern that considers culture as the perfect reference standard. The cleared circles outlined on several coordinates represent the estimated sensitivity and specificity of each of the included studies, and the black circle is a pooled estimate of sensitivity and specificity achieved from the bivariate model. sROC, summary receiver operating characteristic; HRSOC, hierarchal summary receiver operating characteristic.\n\n\nDiscussion\n\nTo the best of our knowledge, this is the first meta-analysis that evaluates the diagnostic accuracy of TB pericarditis using Xpert MTB/RIF. Previously, six systematic reviews had assessed the diagnostic test accuracy of Xpert MTB/RIF for extrapulmonary TB. Chang et al., 201218 (eight studies) and Li Y et al., 201719 (26 studies), analyzed the diagnostic test accuracy of Xpert MTB/RIF for multiple forms of extrapulmonary TB combined. Denkinger et al., 20147 (18 studies), Maynard-Smith et al., 201420 (27 studies), Penz et al., 201521 (37 studies), Sehgal et al., 201622 (24 studies), and Kohli et al., 20185 (18 studies) analyzed the diagnostic test accuracy of Xpert MTB/RIF for specific forms of extrapulmonary TB. However, none of them specifically analyzed the diagnostic accuracy of Xpert MTB/RIF for TB pericarditis.\n\nIn this meta-analysis, the pooled specificity of Xpert MTB/RIF to diagnose pericarditis TB was extremely powerful for the majority of studies using Xpert MTB/RIF to diagnose extrapulmonary TB (0.994 (95% CI: 0.919–1.000), which highlights its effectiveness as a test to rule-in TB pericarditis diagnosis and help clinicians to decide on early TB treatment. This early TB treatment will be able to improve outcomes and reduce mortality for patients23. Interestingly, this study showed better sensitivity and specificity than previous meta-analyses, which studied the use of Xpert MTB/RIF to diagnose various extrapulmonary TB5,7,18–22. This might be because a bivariate model pooled the overall sensitivity and specificity in this study. Thus, the validity of our results were higher than that of the prior meta-analyses. In addition, we limited the diagnosis to TB pericarditis rather than unspecified extrapulmonary TB.\n\nThe sensitivity of Xpert MTB/RIF for TB detection in pericardial fluid varied widely across the studies involved in this meta-analysis. Thus, the pooled sensitivity outcome was not very high (0.676 (95% CI: 0.580–0.759). The paucibacillary characteristics of the TB pericarditis might cause the reduced sensitivity of diagnosis with Xpert MTB/RIF using pericardial fluid24. The detection limit of Xpert MTB/RIF is 131 CFU/ml; any unit lower than this cannot be detected, resulting in negative results25. Hence, this may lead to a false-negative diagnosis of pericarditis TB. Other possible explanation are the variation in the sample size, sampling method, patient characteristics, and settings. In this meta-analysis, 45% of the included studies were conducted in areas with low prevalence. Patients in these areas may present with a paucibacillary disease earlier, which reduce the sensitivity of Xpert MTB/RIF26. Limited sensitivity will hinder the usage of Xpert MTB/RIF for ruling out TB pericarditis, especially when samples are shown to be smear-negative. Meanwhile, LJ culture can detect an organism from as low as 10 CFU/ml to 100 CFU/ml27, resulting in cell culture as the best reference standard for TB detection, especially when a very low number of organisms are involved.\n\nThough the interpretation is not straightforward, the sensitivity, specificity, and AUC under sROC are the fundamental aspects that demonstrate the accuracy of the diagnostic test. Nevertheless, in a clinical setting, NLR and PLR can be essential and exhibit the actual state. In order to identify and rule out target diseases, it is frequently accepted that a PLR above 10 and an NLR below 0.1 signify clinical significance28. In this present study, the PLR and NLR values for Xpert MTB/RIF in the diagnosis of TB pericarditis were 110 and 0.3, respectively. These values indicate that the Xpert MTB/RIF test is reliable in identifying or ruling in pericarditis TB, and is a faster and easier method compared to other tests.\n\nThe strengths of our study were the use of standard protocols from the PRISMA guidelines and the Cochrane Handbook, strict inclusion and exclusion criteria, extraction of data by independent reviewers, and the use of bivariate models to calculate pooled sensitivity and specificity. However, we realized that our study has several limitations. We acknowledge that we might excluded numerous studies of extrapulmonary TB that did not have enough data on TB pericarditis despite comprehensive exploration. Furthermore, this meta-analysis involved a relatively low number of patients (581 eligible patients) due to the limited study of the use of Xpert MTB/RIF to diagnose pericarditis TB. Thus, the result might not reveal the actual state in the population. Further meta-analysis should be conducted when more studies specifically evaluate the use of Xpert MTB/RIF to diagnose pericarditis TB with a larger sample size.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nFigshare: Detailed search strategy. https://doi.org/10.6084/m9.figshare.11974356.v129\n\nFigshare: Review protocol. https://doi.org/10.6084/m9.figshare.1197426030\n\nFigshare: PRISMA checklist for Diagnostic Test Accuracy of Xpert MTB/RIF for Tuberculous Pericarditis: A Systematic Review and Meta-Analysis https://doi.org/10.6084/m9.figshare.11955003.v131\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgments\n\nThis paper and the research behind it would not have been possible without the exceptional support of Faculty of Medicine Universitas Airlangga, Dr. Soetomo General Academic Hospital and Institute of Tropical Disease, Surabaya, Indonesia.\n\n\nReferences\n\nReuter H, Burgess LJ, Doubell AF: Epidemiology of pericardial effusions at a large academic hospital in South Africa. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nUllah I, Javaid A, Masud H, et al.: Rapid detection of Mycobacterium tuberculosis and rifampicin resistance in extrapulmonary tuberculosis and sputum smear-negative pulmonary suspects using Xpert MTB/RIF. J Med Microbiol. 2017; 66(4): 412–418. PubMed Abstract \| Publisher Full Text\n\nYu G, Ye B, Chen D, et al.: Comparison between the diagnostic validities of Xpert MTB/RIF and interferon-γ release assays for tuberculous pericarditis using pericardial tissue. Wilkinson KA, editor. PLoS One. 2017; 12(12): e0188704. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZeka AN, Tasbakan S, Cavusoglu C: Evaluation of the GeneXpert MTB/RIF Assay for Rapid Diagnosis of Tuberculosis and Detection of Rifampin Resistance in Pulmonary and Extrapulmonary Specimens. J Clin Microbiol. 2011; 49(12): 4138–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChang K, Lu W, Wang J, et al.: Rapid and effective diagnosis of tuberculosis and rifampicin resistance with Xpert MTB/RIF assay: A meta-analysis. J Infect. 2012; 64(6): 580–8. PubMed Abstract \| Publisher Full Text\n\nLi S, Liu B, Peng M, et al.: Diagnostic accuracy of Xpert MTB/RIF for tuberculosis detection in different regions with different endemic burden: A systematic review and meta-analysis. Pai M, editor. PLoS One. 2017; 12(7): e0180725. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaynard-Smith L, Larke N, Peters JA, et al.: Diagnostic accuracy of the Xpert MTB/RIF assay for extrapulmonary and pulmonary tuberculosis when testing non-respiratory samples: a systematic review. BMC Infect Dis. 2014; 14(1): 709. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPenz E, Boffa J, Roberts DJ, et al.: Diagnostic accuracy of the Xpert ® MTB/RIF assay for extra-pulmonary tuberculosis: a meta-analysis. Int J Tuberc Lung Dis. 2015; 19(3): 278–84. PubMed Abstract \| Publisher Full Text\n\nSehgal IS, Dhooria S, Aggarwal AN, et al.: Diagnostic Performance of Xpert MTB/RIF in Tuberculous Pleural Effusion: Systematic Review and Meta-analysis. Land GA, editor. J Clin Microbiol. 2016; 54(4): 1133–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarikrishna J, Mohan A: Treatment of tuberculosis pericarditis. J Pract Cardiovasc Sci. 2015; 1(2): 179–181. Publisher Full Text\n\nPasipanodya JG, Mubanga M, Ntsekhe M, et al.: Tuberculous Pericarditis is Multibacillary and Bacterial Burden Drives High Mortality. EBioMedicine. 2015; 2(11): 1634–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChakravorty S, Simmons AM, Rowneki M, et al.: The new Xpert MTB/RIF ultra: Improving detection of Mycobacterium tuberculosis and resistance to Rifampin in an assay suitable for point-of-care testing. mBio. 2017; 8(4): e00812–17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGriffiths C, Barne M, Saxena P, et al.: Challenges of tuberculosis management in high and low prevalence countries in a mobile world. Prim Care Respir J. 2014; 23(1): 106–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAsmar S, Chatellier S, Mirande C, et al.: A novel solid medium for culturing mycobacterium tuberculosis isolates from clinical specimens. J Clin Microbiol. 2015; 53(8): 2566–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDeeks JJ, Altman DG: Diagnostic tests 4: likelihood ratios. BMJ. 2004; 329(7458): 168–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAl Farabi MJ: Detailed Search Strategy. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.11974356.v1\n\nAl Farabi MJ: Review Protocol. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.11974260.v1\n\nAl Farabi MJ: PRISMA Checklist for Diagnostic Test Accuracy of Xpert MTB/RIF for Tuberculous Pericarditis: A Systematic Review and Meta-Analysis. figshare. Online resource. 2020. http://www.doi.org/10.6084/m9.figshare.11955003.v1" }	[ { "id": "67749", "date": "06 Aug 2020", "name": "Pieter A.F.M. Doevendans", "expertise": [ "Reviewer Expertise General cardiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study is combing the available data on a diagnostic test to detect TB as a cause in this case for pericarditis. From the various studies it becomes clear that the specificity is excellent. Sensitivity is however disappointing, maybe due to a low bacterial load. So there is a place for the test in making a fast diagnosis to start early treatment. Yet a negative result does not exclude TB as the possible aetiology.\n\nStudy is well designed well performed and not overstating the technique can be used for rule in. Well done work, well written.\n\nOnly 1 typo on page 8: we might excluded No other corrections are needed at this point in time\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] }, { "id": "67746", "date": "10 Aug 2020", "name": "Mohammad Saifur Rohman", "expertise": [ "Reviewer Expertise Cardiology Translational research : Pharmacogenetic", "Interventional cardiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study is well written with objective and reliable method was used. It is acceptable for scientific standard. It is interesting looking at the result of this study. Which tried to do metanalysis from 9 research in diagnosing tuberculosis related pericardial effusion using Xpert MTB/RIF. The author also already considered bias risk and applicability concern.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-761
https://f1000research.com/articles/9-760/v1	22 Jul 20	{ "type": "Research Article", "title": "Survey to healthcare professionals on the practicality of AI services for healthcare", "authors": [ "Antti Väänänen", "Keijo Haataja", "Pekka Toivanen", "Keijo Haataja", "Pekka Toivanen" ], "abstract": "Background: Artificial Intelligence (AI) is already in use in many fields of healthcare, and utilization of AI in state-of-the art healthcare services is growing, with many studies showing that AI can provide benefits in healthcare processes. However, there is a need for research about healthcare professionals’ willingness to use AI-based services, and their consideration about in which cases AI could provide better healthcare outcomes and reduce healthcare professionals’ workload. Methods: We studied the latest technologies and methods from current healthcare AI services’ end-users to create a survey on healthcare services, including use cases that could be beneficial for healthcare professionals. We focused on studying Information and Communication Technology (ICT) applications and services which utilize modern AI features. The purpose of our survey for healthcare professionals was to provide an analysis of the healthcare services that utilize AI features, which could provide better health outcomes from a healthcare professional and care process point of view. Results: AI features, such as health monitoring, medication management, and connected machines are considered to have high benefit in healthcare workflow. Moreover, we found that the majority (67.8%) of healthcare professionals are willing to use AI for supporting decision making or even providing independent diagnosis. In total, 28.9% of healthcare professionals are willing to use AI in a restricted manner to help professionals in care processes. Only 3.3% of respondents are not willing to use AI-based services at all. Conclusions: Our survey indicated that the willingness of healthcare professionals to use different AI in healthcare technology solutions is high.", "keywords": [ "Artificial Intelligence", "Digital Health", "Healthcare Information Systems", "Automated Diagnoses." ], "content": "Introduction\n\nThe development of Artificial Intelligence (AI) has been very fast in the past decade. AI and robotics are now utilized in almost all business sectors and this will impact the global labor market in the near future1. The following are AI services that have gained the most publicity: robotics, autonomous cars, computer vision applications, and natural language processing services. However, AI is already in use or is emerging on a wide scale in different business sectors2.\n\nWhen considering new models for AI utilization in healthcare processes, it is generally believed that soon AI will automatically give accurate diagnoses, provide and invent new care instructions and new treatments for diseases, and will act as human healthcare professionals but in a much more accurate, precise, and effective way. In this scenario, AI could provide a remarkable advantage to enhance health outcomes and healthcare processes, as well as help caregivers in their demanding work. For example, AI can provide help regarding the shortage of the healthcare workforce and can also provide healthcare resources for aging populations3. In addition, in situations when the global healthcare system is burdened, such as the COVID-19 pandemic, healthcare resources have an increased workload and there is strong demand for AI solutions, which will provide governments and healthcare organizations with the necessary tools to perform actions faster and more efficiently, especially in pandemics4. These are the final goals of utilizing AI in healthcare, but all of these are currently not achievable at the time of writing this article.\n\nTo achieve these goals, we need to study healthcare professionals’ viewpoints concerning current utilization of AI in healthcare and ask professional end-users (clinicians and administrators) of healthcare services about the best use cases where AI could benefit different healthcare processes. Moreover, before we can take full advantage of AI in healthcare, we must tackle ethical and legal obstacles and comply with all regulatory requirements5. Furthermore, metrics to measure benefits and better care outcomes for patients, better care outcomes for healthcare service providers, effectiveness of service, and response time for service queries, must be constructed.\n\nIn this study, we provide the results of a survey conducted to healthcare professionals about use of AI in healthcare practices. Based on the results we provide some evaluation of AI features that could be widely accepted by healthcare professionals. Moreover, we present new ideas that will be used in our future research work and insights for a novel upcoming healthcare AI-service platform. We hope that this novel AI-based healthcare solutions platform will be able to support healthcare service providers, caregivers, and customers in a more effective way compared with traditional healthcare IT services.\n\n\nMethods\n\nThe aim of the survey was to evaluate utilization and acceptance of different AI-based services of healthcare professionals working in the healthcare sector. We also provide a comparative analysis of our survey and an earlier survey6, where the adoption of AI services by healthcare professionals and public users were studied.\n\nThe main areas of AI-based healthcare are clinical decision making, healthcare interventions, automatic care or healthcare process recommendations, patient administration, and patient monitoring. For this study, we searched the literature for research and utilization of AI technology in the healthcare sector. Based on our search, we selected healthcare services that included AI technologies in natural language processing, neural networks and deep learning, computer vision or robotics, rule-based expert systems, and physical robots7,8. This selection was made on the basis that there are many studies concerning these AI technologies and they are commonly in use in the healthcare sector. Therefore, healthcare services utilizing AI that were used as examples in our survey are as follows: robot-assisted surgery, virtual assistants for nursing and consultation, assistant for administration and workflow, fraud detection, dosage error reduction, connected machines, clinical trial participation, preliminary diagnosis, image diagnosis, cybersecurity, medication management, health monitoring, and drug creation.\n\nThe survey was designed to analyze the most suitable services and features to support healthcare professionals and administrators in their work, as well as which features and services could provide best care outcomes for the patients. The survey contained three parts: part 1 asked for demographic characteristics of respondents; part 2 asked general questions concerning technology and AI; and part 3 asked about an individual’s acceptance of AI technology and its advantages for certain use cases. Question types were single selection, multi-selection, open number value, and 5-step Likert Scale9. The survey can be found in full in the Extended data.\n\nThe survey was created using Google Forms. A link to the survey was sent to healthcare professionals by informing healthcare professionals in our own network in Finland via Facebook and using SurveyCircle scientific survey service10. No individual requests to complete the survey were sent. The survey was accessible from December 19, 2019 to February 25, 2020.\n\nThe survey was designed to provide data in a formal structured manner to support our analysis in an optimal way and enable us to smoothly continue with our practical implementation efforts of future research work ideas.\n\nWe used basic statistical analysis tools from Excel Analysis ToolPak11, presenting mean values and standard deviation for each question, and analyzed respondents’ staff group, work experience, and age against AI technology adoption. We analyzed how respondents utilize IT technology in their work, as well as their willingness to adopt new healthcare services that use AI technology. Finally, we compared our survey results to the survey results conducted by YouGov Research in November 20166 to the general public in 12 countries across Europe, the Middle East, and Africa (n=12000). Our aim was to find out if our survey results correlate with the survey results from YouGov Research.\n\nEthical approval for the study protocol and survey was waived by the University of Eastern Finland Research Ethics Committee. This waiver of approval was provided since the study did not contain any of the following: research deviates from principle of informed consent; research involves intervening in the physical integrity of participants; research is on minors under the age of 15 years; research exposes participants to strong stimuli; research involves risk of causing mental harm to participants; and research involves threat of safety to participants.\n\nParticipants were informed about the aim of the questionnaire and that this information would be used in research, which was shown at the beginning of the questionnaire. Completion of the questionnaire was taken as consent to participate.\n\n\nResults\n\nWe created an Internet survey to examine the usage, usability, and end-user acceptance of AI-based healthcare services by healthcare professionals. We received a total of 125 responses. Of these, only 121 responses were 100% complete and therefore only these responses were analyzed.\n\nThe study population gender distribution was female 63.6% (77), male 35.6% (43), and other 0.8% (1). Mean age and mean work experience was not able to be calculated accurately because of the age and experience questions did not contain exact age or years of experience. Respondents staff group distribution was as follows: nursing staff, 48.8% (59); office and administration staff, 14.9% (18); research staff, 14.0% (17); physicians, 14% (17); social work, 5.0% (6); and maintenance staff, 3.3% (4). The demographics of the study population are shown in Figure 1.\n\nThe survey measured healthcare professionals’ opinions about how AI technology will be able to provide enhancement to care processes and functions, and if AI can replace some activities or tasks that are traditionally done by healthcare professionals or traditional healthcare ICT services. The survey results were analyzed by each question and with basic statistical analysis tools (mean values and standard deviation).\n\nIn the survey we asked about the current use of different digital services and features among healthcare professionals (question G1). When analyzing the results, we noticed that respondents stated that the most currently used ICT services listed in the survey were for drug discovery (31.4%), medical data security (28.1%), medical diagnosis (24%), signal analysis (23.1%), and finding participants to take part in clinical trials (23.1%). The least used services were robot surgery (6.6%) and fraud detection (9.1%). In total, 19% of professionals did not use any of the listed digital services in their work (Figure 2).\n\nFor the utilization of these ICT services, 19% of respondents did not use any of the services listed in G1. For the technology usage level (question G3), we found that 20% of the respondents used these services daily and 24% at least once per week (Figure 3).\n\nWe also asked health professionals which healthcare ICT services and features were considered the most beneficial in their work and/or care processes (question G2). There was the possibility to select multiple technologies. The respondents reported that the following were the most beneficial/used: health monitoring (43.8%), medication management (32.2%), connected machines (27.3%), dosage error reduction (23.1%), and signal analysis (18.2%). The least beneficial services for healthcare professionals were cybersecurity (8.3%) and fraud detection (9.1%). In total, 10.7% of respondents do not want to use any of the listed services in their work (Figure 4).\n\nOne of the questions in the survey was for healthcare professionals to consider the advantages of AI-based services (question G4; Figure 5). The response was a 4-step Likert scale from 0 to 4, where 0 is “Do not agree at all” and 4 is “I fully agree”. The results showed that three most considered advantages of AI by healthcare professionals were “Simple and repeating tasks can be performed by AI/robots” (mean score 3.0), “AI can provide help in healthcare professionals workload” (mean score 3.0), and “AI can provide easier access for more people/patients to healthcare services” (mean score 3.0). These services have also lowest standard deviation (0.86 – 0.95). Other lesser considered advantages were “AI can give better treatment recommendations” (mean score 2.3) and “AI can perform surgery more accurately than healthcare professionals” (mean score 2.3). When analyzing the average scores from all replies there is an indication that healthcare professionals mostly consider AI-based services and features in healthcare to have advantages.\n\nLikert scale from 0 to 4, where 0 is “Do not agree at all” and 4 is “I fully agree”.\n\nWe also asked what disadvantages healthcare professionals consider AI to have when used in healthcare (question G5, Likert scale as for question G4; Figure 6). The highest considered disadvantage was given for “AI or robots cannot be in contact with patients. Patients need human interaction when providing healthcare services.” (mean score 2.8), “Too complicated for healthcare professionals to access AI technology solutions” (mean score 2.1) and “AI does not provide accurate results for decision making. Only healthcare professionals can make right decisions.” (mean score 2.1) The lowest value was given to “I can see only disadvantages when using AI or robotics in healthcare.” (mean score 1.2). The results of this question show that some healthcare professionals express their concern that AI should not have contact with patients without human interaction and the final decision in healthcare processes should be kept in human hands. We can also see that healthcare professionals consider that AI-based solutions bring advantages rather than disadvantages.\n\nLikert scale from 0 to 4, where 0 is “Do not agree at all” and 4 is “I fully agree”.\n\nWe asked healthcare professionals about willingness to use healthcare services that utilize AI (questions A1-A15). Respondents could evaluate each service independently with a 4-step Likert scale between 0 to 4, with 0 as “Not willing” and 4 as “Very willing”. A mean utilization score of 2.8 was given to services used for automatic health monitoring, providing enhanced data security, providing fraud detection, providing medical risk prediction, and conducting health assessment questionnaires. These services also have the lowest standard deviation (0.80 – 0.90). Lowest utilization scores were given to services such as AI-assisted major surgery (mean score 1.9), AI giving diagnosis of patient (2.3), and AI providing instant healthcare advice for the patient (2.3). Overall results are shown in Figure 7.\n\nLikert scale between 0 to 4, with 0 as “Not willing” and 4 as “Very willing”.\n\nFinally, we asked healthcare professionals how they are to have AI make decisions or give recommendations in healthcare processes (question G6). The responses showed that most healthcare professionals are willing to use AI in healthcare in all possible cases with the final decision made by humans (65%) or AI to give decisions without human interaction (3%). Also 29% of respondents consider that AI can ease healthcare work and processes, but only by giving recommendations. Only 3% of respondents consider that AI is not suitable for healthcare.\n\nWe analyzed how respondents’ staff group and age group correlate to the willingness to use AI technology. Social work and maintenance staff had a total 10 respondents each and were therefore combined. We found that there was only minor deviation among different staff groups supporting AI utilization (Figure 8).\n\nBlue, “AI can be used in all possible cases, but humans should always make final decision”; orange, “AI can ease clinical work and clinical processes but only with restricted matter (only by giving recommendations)”; black, “AI is not suitable for healthcare”; grey, “AI can be used in healthcare and can independently provide diagnoses and decisions for care”.\n\nSecondly, we analyzed age groups which have over 15 respondents. Age groups (55–64 years and over 65 years) had total 9 respondents and those were combined. We found that there was only minor deviation among different age groups supporting AI utilization (Figure 9).\n\nBlue, “AI can be used in all possible cases, but humans should always make final decision”; orange, “AI can ease clinical work and clinical processes but only with restricted matter (only by giving recommendations)”; black, “AI is not suitable for healthcare”; grey, “AI can be used in healthcare and can independently provide diagnoses and decisions for care”.\n\nThirdly, we analyzed work experience from the groups that have over 15 respondents. Work experience groups (55 – 64 years and over 65 years) had total 9 respondents and those are combined to last pie chart. We found that there was only minor deviation among different work experience groups supporting AI utilization (Figure 10).\n\nBlue, “AI can be used in all possible cases, but humans should always make final decision”; orange, “AI can ease clinical work and clinical processes but only with restricted matter (only by giving recommendations)”; black, “AI is not suitable for healthcare”; grey, “AI can be used in healthcare and can independently provide diagnoses and decisions for care”.\n\n\nDiscussion\n\nFrom the survey results, we found out that the AI technology that respondents consider to be most useful for healthcare work processes (question G2) are services that provide health monitoring, medication management, and networked devices and databases which can exchange data between each other. In addition, the three most currently used healthcare IT services by respondents are drug discovery, medical data security, and medical diagnosis (question G1).\n\nWe compared our research results to an earlier survey conducted by YouGov Research6. We found similarities in perceived advantages of AI between the two surveys. In the YouGov survey, the top two advantages that respondents reported were “Healthcare would be easier and quicker for more people to access” and “Advanced computers/robots can make diagnosis faster and more accurately”. From our survey, the top three perceived advantages identified by healthcare professionals were “AI can provide easier access for more people/patients to healthcare services”, “AI can provide help in healthcare professionals workload”, and “Simple and repeating tasks can be performed by AI/robots”.\n\nWhen we compared disadvantages between surveys, we found that the top three disadvantages reported in our survey were “AI or robots cannot be in contact with patients. Patients need human interaction when providing healthcare services”, “Too complicated for healthcare professionals to access AI technology solutions”, and “AI does not provide accurate results for decision making. Only healthcare professionals can make right decisions”. The disadvantages reported by the YouGov survey were “People need the human touch when it comes to their healthcare”, “If something unexpected is found during surgery or in a test, I do not trust robots to make decisions on what to do”, and “Only a human healthcare professional can make the right decisions”. Therefore, a correlation in perceived disadvantages by both groups of respondents can be seen.\n\nWhen analyzing the demographic data of respondents and survey responses, we concluded that age and staff group among healthcare professionals does not have significant effect on adoption of AI features or willingness to use healthcare services utilizing AI methods. There was also an indication from the survey results that respondents of a younger age and shorter work experience in the healthcare sector correlated negatively with willingness to adopt AI technology in healthcare.\n\nIn addition, we found that willingness to use novel features and services utilizing AI by healthcare professionals is high when novel features are used to help healthcare professionals in their decision making or work processes. Perceived disadvantages when using AI services mainly concerned loss of human interaction when robotics is used in healthcare and unwillingness to let AI make clinical decisions.\n\nOverall, we conclude that using AI in healthcare is accepted by healthcare professionals and there are many AI enabled features and services where there could be of high potential in healthcare. However, developers and researchers of these novel AI services must keep in mind that the perceived disadvantages should be solved and human decision making and human interaction in these novel services is necessary, until these new services gain user acceptance and adoption in the healthcare sector. From the YouGov survey, these same acceptance criteria also apply when considering healthcare AI service adoption within the general public.\n\nIn our future research work we will evaluate more results of this survey to find the most effective combination of AI-services and technologies that could provide the best benefits for healthcare work processes, health outcomes, and preventive health processes. We will focus on providing services that could be easily adopted by healthcare professionals and the general public, and that should provide imminent enhancement in care processes. Moreover, we will provide a comparative study of healthcare technologies where AI is used as a key element and evaluate the effectiveness of these technologies in the field of healthcare. Finally, we will propose a technology platform where these studied AI technologies could be easily adopted by healthcare professionals and their customers or patients.\n\n\nData availability\n\nOpen Scientific Framework: Survey to healthcare professionals on the practicality of AI services for healthcare, https://doi.org/10.17605/OSF.IO/674ZM12.\n\nOpen Scientific Framework: Survey to healthcare professionals on the practicality of AI services for healthcare, https://doi.org/10.17605/OSF.IO/674ZM12.\n\nThis project contains the following extended data:\n\n- Survey questions in English and Finnish.\n\n- Survey questions in table format (Tables 1–3)\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\nWisskirchen G, Biacabe BT, Bormann U, et al.: Artificial Intelligence and Robotics and Their Impact on the Workplace. IBA Global Employment Institute 2017; 2012–2017. Reference Source\n\nBughin J: Artificial Intelligence the Next Digital Frontier. McKinsey Global Institute Discussion Paper. 2017. Reference Source\n\nMeskó B, Hetényi G, Győrffy Z: Will Artificial Intelligence Solve the Human Resource Crisis in Healthcare? BMC Health Serv Res. 2018; 18(1): 545. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcCall B: COVID-19 and Artificial Intelligence: Protecting Health-Care Workers and Curbing the Spread. Lancet Digit Health. 2020; 2(4): e166–e167. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFood and Drug Administration: Proposed Regulatory Framework for Modifications to Artificial Intelligence/Machine Learning (AI/ML)-Based Software as a Medical Device (SaMD)-Discussion Paper. 2019. Reference Source\n\nPWC: Why AI and Robotics Will Define New Health. referred 12.2.2020. Reference Source\n\nDavenport T, Kalakota R: The Potential for Artificial Intelligence in Healthcare. Future Healthc J. 2019; 6(2): 94–98. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReddy S, Fox J, Purohit MP: Artificial Intelligence-Enabled Healthcare Delivery. J R Soc Med. 2019; 112(1): 22–28. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nScienceDirect: Likert Scale. referred 20.3.2020. Reference Source\n\nSurveyCircle. 2019. Reference Source\n\nMicrosoft: Use the Analysis ToolPak to perform complex data analysis. referred 15.3.2020. Reference Source\n\nVäänänen A: Survey to healthcare professionals on the practicality of AI services for healthcare. 2020. http://www.doi.org/10.17605/OSF.IO/674ZM" }	[ { "id": "72922", "date": "15 Oct 2020", "name": "Parag Chatterjee", "expertise": [ "Reviewer Expertise Artificial Intelligence", "eHealth" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work is aimed at an interesting aspect of Artificial Intelligence in healthcare — the acceptability and opinions of the key stakeholders of the medical and healthcare system. The survey has been conducted in a well-structured manner, and the results are illustrated and analyzed well. However, considering the size of the cohort of this survey, a brief introduction and background of the participants would have been useful. Since some parts of the survey are quite subjective, it is important to explain the healthcare system and intrinsic relationships as well, where these insights from the survey hold true. The use of the words 'ICT Technology' is redundant, since ICT itself is 'Information and Communication Technology'. Based on the survey outcomes, it is interesting to see a high level of acceptance in healthcare professionals towards Artificial Intelligence in healthcare.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "81941", "date": "01 Apr 2021", "name": "Charlotte Blease", "expertise": [ "Reviewer Expertise Philosophy of medicine", "informatics", "future of the health professions" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nUnfortunately, there are some serious problems with this survey. The abstract does not tell us anything about the sample or response rate. We later learn that the survey is drawn from a convenience sample drawn from a local network in Finland. There is therefore no assurance about the representativeness of health professionals in Finland. Indeed, the respondents include a variety of professions - physicians, nurses, admin staff, maintenance staff. Therefore we do not have a clear understanding of the attitudes/opinions about a particular demographic group of health professionals in Finland. The variety of perspectives limits the possibility of drawing robust inferences. In the Introduction, there is no adequate literature review, nor a clear outline of the objectives in light of the background literature. We also need some commentary on whether it is is useful to survey health professionals on these questions. i.e. why would we expect health professionals to have a clear understanding of the applications of AI/ML to healthcare? This was not addressed. What do the investigators consider the utility of the results of the survey to be? Educational interventions? The authors do not adequately address the limitations of the survey.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "81945", "date": "19 Apr 2021", "name": "Lorenzo Faggioni", "expertise": [ "Reviewer Expertise Artificial intelligence", "Medical imaging", "Radiology", "Imaging informatics" ], "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 interesting manuscript reporting findings from a local survey on the expectations of healthcare professionals about the use of AI in healthcare. However, it has substantial flaws that should be addressed, including the following:\nThe survey findings and demographic data of the survey respondents were reported and illustrated in detail, but apparently without any sort of statistical analysis (which could allow drawing rigorous conclusions based on statistical significance). Hence, I would strongly encourage the authors to analyze their data using inferential statistical methods (including p-values, confidence intervals comparisons, etc.), in order to find objective associations and/or correlations in their findings. For instance, it would be very important to know if there were any statistically significant correlations between the replies of survey participants and their age, job/specialty and work experience.\n\nThe entire Discussion section should be focused on systematically comparing the study findings with those of the relevant existing literature. Basically, the only comparison was made with the YouGov survey, yet there are several surveys published in the literature regarding the awareness of healthcare professionals about AI and their expectations (either positive or negative) about its usability. Useful references include (but are not limited to) those listed in the References list attached to this review.1,2,3,4,5,6,7,8,9,10,11\n\nI would mention the rather small study sample (N=121 complete responses) as a potential limitation of the study and comment on whether such limitation can impact the study findings and conclusions.\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? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-760
https://f1000research.com/articles/8-1822/v1	29 Oct 19	{ "type": "Method Article", "title": "A hands-on introduction to querying evolutionary relationships across multiple data sources using SPARQL", "authors": [ "Ana Claudia Sima", "Christophe Dessimoz", "Kurt Stockinger", "Monique Zahn-Zabal", "Tarcisio Mendes de Farias", "Ana Claudia Sima", "Christophe Dessimoz", "Kurt Stockinger", "Monique Zahn-Zabal" ], "abstract": "The increasing use of Semantic Web technologies in the life sciences, in particular the use of the Resource Description Framework (RDF) and the RDF query language SPARQL, opens the path for novel integrative analyses, combining information from multiple sources. However, analyzing evolutionary data in RDF is not trivial, due to the steep learning curve required to understand both the data models adopted by different RDF data sources, as well as the SPARQL query language. In this article, we provide a hands-on introduction to querying evolutionary data across multiple sources that publish orthology information in RDF, namely: The Orthologous MAtrix (OMA), the European Bioinformatics Institute (EBI) RDF platform, the Database of Orthologous Groups (OrthoDB) and the Microbial Genome Database (MBGD). We present four protocols in increasing order of complexity. In these protocols, we demonstrate through SPARQL queries how to retrieve pairwise orthologs, homologous groups, and hierarchical orthologous groups. Finally, we show how orthology information in different sources can be compared, through the use of federated SPARQL queries.", "keywords": [ "Orthology", "Comparative Genomics", "Sequence Homology", "Resource Description Framework (RDF)", "SPARQL" ], "content": "Introduction\n\nGene classification based on evolutionary history is essential for many aspects of comparative and functional genomics - reviewed in (Gabaldón & Koonin, 2013); (Glover et al., 2019). On the one hand, evolutionary relations are often described as binary relations. Two genes that share a common ancestor are defined as homologs. We can classify homologs into orthologs, which originate from a speciation event; paralogs, which originate from a gene duplication; and xenologs, which originate from horizontal gene transfer (Fitch, 1970). On the other hand, Hierarchical Orthologous Groups (HOGs) are hierarchical clusters of corresponding genes where each level in the hierarchy refers to a common ancestral gene at a taxonomic level of reference (Altenhoff et al., 2013). Identifying orthologs and HOGs is valuable in several contexts such as gene function inference, gene evolution dynamics and comparative genomics.\n\nTo query and interoperate biological databases, Semantic Web Technologies are being increasingly adopted, in particular the use of the Resource Description Framework (RDF) and SPARQL protocol and RDF query language (SPARQL Query Language for RDF, n.d.). However, despite the progress they have enabled in several fields, particularly in the life sciences (Duek et al., 2018), (Iyappan et al., 2016) (Williams et al., 2012), there are still significant challenges that limit their use for the larger scientific community. In particular, analysing evolutionary relationship data in RDF poses the following challenges:\n\n1) complex data models - for example, while storing data in a hierarchical structure (HOGs) results in significant performance benefits for common analyses, such as computing orthologs of a specific gene in a different model organism, the hierarchy also results in requiring advanced knowledge of the SPARQL language (in particular, recursivity) in order to benefit from the RDF representation of HOGs. In this article, we present a series of hands-on examples, in increasing order of complexity, to familiarise the reader with the basic concepts needed to query evolutionary relationships in orthology databases.\n\n2) heterogeneous data models - understanding the data model of a single orthology database might not be sufficient in general, since different databases have made different design decisions. We help overcome this challenge by depicting how the following major Orthology Databases structure their data in RDF, as well as how they can be queried using SPARQL: the Orthologous MAtrix (OMA) (Altenhoff et al., 2018), the European Bioinformatics Institute (EBI) RDF platform (Jupp et al., 2014), the Database of Orthologous Groups (OrthoDB) (Zdobnov et al., 2017) and the Microbial Genome Database (MBGD) (Uchiyama et al., 2018).\n\n3) overhead of integration into existing analysis pipelines. The limited rate of adoption of Semantic Web Technologies can be explained by the reluctance of bioinformaticians to change their existing workflows in order to accommodate new data formats based on the RDF framework. For example, retrieving orthology information using public SPARQL endpoints instead of the more traditional file-based data exchange or full database dumps. A SPARQL endpoint is an access point for receiving and processing SPARQL protocol requests. In this article, we show through concrete examples that integrating the results of SPARQL queries into existing analyses is a straightforward task - more specifically, we show how to transform the results into regular Pandas dataframes in Python. Furthermore, we provide an accompanying Jupyter notebook where all the examples presented in this article can be directly tested and further refined.\n\nThis article has several goals:\n\n(1) Understanding Orthology Data Models. Become familiar with how evolutionary relationships are represented in RDF across several databases. Learn about the data modelling decisions: common points as well as differences between these sources to support the choice of one or more of them for a given analysis.\n\n(2) Understanding how to query orthology data using SPARQL. To this end, we extend the introduction and examples in (Chiba et al., 2015) (de Farias et al., 2017), while also covering multiple, distributed orthology data sources.\n\n(3) Integrating external sources. Leverage connections to other external bioinformatics resources that make their data available in public SPARQL endpoints based on cross-references. In particular, learn about the role of UniProt cross-references as a bridge between different data sources in integrative analyses.\n\nIn addition, we show how to use SPARQL to make meta-analyses combining multiple orthology databases. For instance, for a given gene, which are the orthologs in a given database which are not present in another one? Finally, we show how to leverage SPARQL aggregations in order to get useful statistics about orthology data available in the sources.\n\nFinally, learn how to leverage SPARQL results in downstream analyses by converting them to Pandas dataframes. This is illustrated through a series of hands-on exercises in the accompanying Jupyter Notebook (exercises provided in Python).\n\nThe protocols presented in this article are aimed at bioinformaticians who are already familiar with the basics of SPARQL and wish to learn how orthology data can be integrated in their research analyses programmatically, through the use of (federated) SPARQL queries.\n\n\nMaterials\n\nIn the following paragraphs, we briefly describe the orthology databases considered in this article.\n\nOrthoDB (Zdobnov et al., 2017) contains orthologous genes along with evolutionary and functional annotations. It relies on HOGs to enable different orthology information resolutions with regards to more closely related species. The 2018 OrthoDB version covers thousands of eukaryotes, prokaryotes, and viruses. OrthoDB data is available in RDF through the public SPARQL endpoint at https://sparql.orthodb.org/sparql. We note here that the timeout for the public SPARQL endpoint is limited to 100 seconds - more precisely, queries with longer estimated execution time will not be allowed to run.\n\nMBGD (Uchiyama et al., 2018) is a comparative genomics database that contains orthology information about bacteria, archaea and unicellular eukaryotes. The 2018 MBGD version has more than six thousand genomes. The MBGD SPARQL endpoint is available online at http://mbgd.genome.ad.jp/sparql/.\n\nOMA (Altenhoff et al., 2018) provides orthologous gene inferences covering all three domains of life: Archaea, Bacteria, and Eukarya. Although mainly focusing on orthology information, OMA also provides paralogy information (i.e. genes related by duplication). Other homology information is not explicitly available but might be manually or automatically extracted from HOGs (de Farias et al., 2017). The 2018 OMA version has 2167 species and can be queried through the SPARQL endpoint at https://sparql.omabrowser.org/lode/sparql/.\n\nEBI is one of the largest bioinformatics resource providers in Europe (Brooksbank et al., 2014). The EBI RDF platform includes pairwise orthologous genes information from Ensembl database (Zerbino et al., 2018). The SPARQL endpoint to access the EBI data is available at https://www.ebi.ac.uk/rdf/services/sparql. For further details, see https://www.ebi.ac.uk/rdf/documentation/ensembl/.\n\nWe group the aforementioned databases based on the orthology information type they provide as follows:\n\nHierarchical Orthologous Groups (HOGs). The three data sources that provide evolutionary relationship data in RDF to represent HOGs are OrthoDB, MBGD and OMA. Although the RDF data models of MBGD and OMA both rely on the ORTH ontology (Fernández-Breis et al., 2016), they use different ORTH versions. However, SPARQL queries running over either of the two sources can be formulated in a very similar manner. In the case of OrthoDB, data are organised according to their own internal data model, while also providing cross-references to the UniProt RDF store.\n\nHomologous groups are sets of homologous genes without any hierarchical grouping (“flat”). All members are homologous to all other members, with no distinction of paralogy or orthology. However, each homologous group can still be associated with a taxonomic level, which indicates to which species clade its members belong. Example of orthology databases from which we can extract these homologous groups are OMA, OrthoDB and MBGD.\n\nPairwise orthology. Apart from the aforementioned orthologous groups, evolutionary data can also be provided in the form of pairwise orthologous genes. Among the sources that provide this type of information in RDF, we consider in this article EBI, OMA and MBGD.\n\n\nData models\n\nIn this section we provide a brief introduction to the data models of the orthology databases considered in this article, in order to facilitate the understanding of the SPARQL queries presented in the Protocol Section.\n\nFigure 1 illustrates a few of the members of a HOG, the main data structure in MBGD. In particular, this MBGD cluster has the identifier 28799. Members of an MBGD orthologous cluster can be either genes, domains or other clusters. These nested orthologous clusters are built at specific taxonomic levels in the hierarchy. For example, the cluster highlighted in blue in Figure 1 was built at taxonomic level 32, Myxococcus. The hierarchy needs to be traversed in order to reach genes, such as mxa:PL1911 that is highlighted in red in Figure 1, or domains (sub-gene level) which belong to an orthologous cluster at a given taxonomic level.\n\nA cluster can consist of genes, domains (sub-genes) or further nested orthologous clusters. Multiple levels of the hierarchy may need to be traversed recursively in order to reach a given orthologous gene. For example, the gene mxa:PL1911 (highlighted in red) can be reached through the member orthologous cluster 2018-01_tax32_8537 (shown in blue). This can be achieved in SPARQL through a recursive graph pattern, using the hasHomologous* property path - a graphical abstraction of the RDF representation is provided in Figure 2.\n\nThe RDF model is more suitable for representing such hierarchical data structures than the relational data model (Sima et al., 2019) given that RDF is a graph data model. Moreover, querying orthology RDF data can benefit from SPARQL 1.1 recursive graph patterns such as property paths1. The main construct in SPARQL required to retrieve the orthologous genes of a gene of interest X will then be the following recursive pattern:\n\n?hog_cluster a orth:OrthologsCluster.\n\n?hog_cluster orth:hasHomologous* ?gene_X.\n\n?hog_cluster orth:hasHomologous* ?orthologous_gene_Y.\n\nFor example, we can replace ?gene_X with the URI of the human Hemoglobin Subunit Beta (HBB) gene, namely: <http://mbgd.genome.ad.jp/rdf/resource/gene/hsa:HSA_4504349>, which would enable retrieving all orthologs of human HBB through the ?orthologous_gene_Y variable. The asterisk () following the “orth:hasHomologous” property indicates that this property should be matched recursively.\n\nA graphical abstraction of the RDF data structure in MBGD is given in Figure 2. SPARQL queries can be formulated by following the direction and labels of arrows in order to formulate triple patterns. For example, to retrieve all genes (i.e. instances of the orth:Gene class) of a given HOG, we can follow the graph structure from root to leaf members by performing the query in the following code fragment. In other words, the ?gene1 variable values illustrated as the left-side member in the cluster ?hog_cluster.\n\nEdges are RDF properties and nodes are either classes or variables. The terms preceded by a question mark (e.g. ?gene1) represent variables assigned with either zero or more literals or URIs. Dashed edges illustrate the orth:hasHomologous property that can be stated zero or more times, recursively. URI prefixes were omitted. MBGD is gene-centric and taxonomic ranges where HOGs are built are not directly available in RDF - in some cases these can be extracted from the cluster URI (e.g. http://mbgd.genome.ad.jp/rdf/resource/cluster/2018-01_tax32_8537 corresponds to taxonomic identifier 32, Myxococcus). By contrast, the taxonomic information per gene entry is richer in MBGD than in OMA, including explicit Superkingdom and Phylum information. Example SPARQL queries based on this graph abstraction are provided in the Protocols, as well as in the accompanying Jupyter Notebook. The pairwise orthology information is not directly available (e.g. through an RDF property), but can be extracted from the Orthologs Cluster (to highlight this, the “isPairwiseOrthologous” is shown in green with a dotted arrow).\n\nPREFIX orth: <http://purl.org/net/orth#>\n\nPREFIX cluster-id:<http://mbgd.genome.ad.jp/rdf/resource/cluster/>\n\nSELECT ?hog_cluster ?gene1 WHERE {\n\nVALUES ?hog_cluster {cluster-id:2018-01_default_28799}\n\n?hog_cluster a orth:OrthologsCluster.\n\n?hog_cluster orth:hasHomologous ?gene1.\n\n?gene1 a orth:Gene. }\n\nThis SPARQL query will retrieve all the genes in the MBGD Hierarchical Orthologous Group represented with the identifier 28799.\n\nSimilarly, the HOG structure in OMA is abstracted in Figure 3. Both figures can be used as a guide in formulating SPARQL queries, by following the directions of the arrows in order to formulate triple patterns. Since both the MBGD and the OMA models rely on the ORTH ontology (Fernández-Breis et al., 2016), the two graph structures are very similar and therefore SPARQL queries can be formulated with only minor differences for both data sources.\n\nEdges are RDF properties and nodes are either classes or variables. The terms preceded by a question mark (e.g. ?protein1) represent variables. Dashed edges illustrate the orth:hasHomologousMember property that can be stated zero or more times, recursively. OMA is protein-centric, however the corresponding genes that encode the proteins are also available in RDF through the \"is encoded by\" property (a cross-reference to Ensembl identifiers is also provided). Furthermore, the taxonomic ranges where HOGs were built are asserted through the \"hasTaxonomicRange\" property. The pairwise orthology information is not directly available (e.g. through an RDF property), but can be extracted from the Orthologs Cluster (to highlight this, the “isPairwiseOrthologous” is shown in green with a dotted arrow). Note: URI prefixes were omitted.\n\nFigure 4 illustrates the data model of the portion of the EBI RDF graph describing orthology information. In contrast to OMA and MBGD, EBI only provides pairwise orthologous genes.\n\nEdges are RDF properties and nodes are either classes or variables. The terms preceded by a question mark (i.e. ?) represent variables assigned with either zero or more literals or URIs. As opposed to the RDF representations in OMA and MBGD, here the pairwise orthology is explicitly asserted through the \"is orthologous to\" property (more precisely, http://semanticscience.org/resource/SIO_000558). However, there is no information available regarding orthologous clusters. Moreover, the Gene class here is in fact the OBO (not ORTH) class, i.e. http://purl.obolibrary.org/obo/SO_0000704. Instances of these genes can be specified either through their cross-reference to UniProt (the http://rdf.ebi.ac.uk/terms/ensembl/DEPENDENT property) or directly through their ENSEMBL identifier, by fixing the value of ?gene to the concatenation of http://rdf.ebi.ac.uk/resource/ensembl/ and the corresponding Ensembl identifier. Finally, the taxonomic identifiers are provided via instances of the BioSource class, http://www.biopax.org/release/biopax-level3.owl#BioSource.\n\nFigure 5 illustrates the structure of Orthologous Groups in the OrthoDB RDF. Here, genes are direct members of OrthoGroups built at a given taxonomic level (Clade), e.g. Cyanobacteria. We mention that OrthoDB provides richer information in RDF, including sequence length, number of exons for gene members, as well as evolutionary rates, functional category and others for orthologous groups (for more details see Extended data).\n\nEdges are RDF properties and nodes are either classes or variables. The terms preceded by a question mark (i.e. ?) represent variables assigned with either zero or more literals or URIs. Genes are direct members of OrthoGroups built at a given taxonomic level (Clade), e.g. Cyanobacteria, available through the ogBuiltAt property. The cross-references to UniProt (as well as Ensembl and Entrez) are available through a 2-triple pattern (for examples see Protocols section).\n\n\nProtocols\n\nIn this section, we provide four protocols to (i) retrieve pairwise orthologs through SPARQL queries from EBI, OMA, MBGD, as well as (ii) homologous groups from OMA, MBGD and OrthoDB (iii) restrict the search to a given taxonomic level (iv) perform meta-analyses across multiple data sources providing orthology information, as well as aggregations using the entire data available in a given source. All protocols presented below are included in the accompanying Jupyter notebook.\n\nFor the sake of simplicity, genes are identified with either their Ensembl identifiers or their cross-reference to the UniProt accession number. In this article, we assume the reader already knows the UniProt primary accession number of the searched gene. In general, this number can be found by searching for the corresponding gene name in the UniProt webpage, for example, “HBB” (i.e. “hemoglobin subunit beta”). The UniProt protein identifier in RDF is a Uniform Resource Identifier (URI) composed of the UniProt accession number (e.g. P68871) appended to the UniProt namespace prefix: http://purl.uniprot.org/uniprot/. For instance, in the case of the human HBB gene, the URI identifier is http://purl.uniprot.org/uniprot/P68871.\n\nIn this protocol we illustrate the basic task of retrieving the pairwise orthologs of a given gene, for example the HBB (Hemoglobin subunit beta) human gene. This is illustrated on the three orthology databases that provide pairwise orthology information in RDF: EBI, OMA and MBGD. The corresponding SPARQL queries to retrieve the pairwise orthologs can be formulated as shown below. We note that the resulting orthologs are also provided using their “clickable” cross-reference link to UniProt. This can directly be used to find out more information about the resulting genes (e.g. name, location, expression) and has the added advantage that results originating from different orthology databases can then be compared against each other.\n\na) Retrieving EBI pairwise orthologs\n\nThe following code fragment depicts a SPARQL query to retrieve pairwise orthologs of the human HBB gene from Ensembl dataset at the EBI RDF platform. To execute this query, copy and paste the it into the Web interface of the EBI SPARQL endpoint at https://www.ebi.ac.uk/rdf/services/sparql.\n\nPREFIX obo: <http://purl.obolibrary.org/obo/>\n\nPREFIX sio: <http://semanticscience.org/resource/>\n\nPREFIX ensembl: <http://rdf.ebi.ac.uk/resource/ensembl/>\n\nPREFIX ensemblterms: <http://rdf.ebi.ac.uk/terms/ensembl/>\n\nSELECT DISTINCT ?gene_uniprot_uri ?ortholog_uniprot_uri {\n\nVALUES(?gene_uniprot_uri){(<http://purl.uniprot.org/uniprot/P68871>)}\n\n?gene sio:SIO_000558 ?ortholog . # « is orthologous to »\n\n?gene obo:RO_0002162 ?taxon . # « in taxon »\n\n?ortholog obo:RO_0002162 ?ortholog_taxon.\n\n?ortholog ensemblterms:DEPENDENT ?ortholog_uniprot_uri.\n\n?gene ensemblterms:DEPENDENT ?gene_uniprot_uri.\n\nFILTER(?taxon != ?ortholog_taxon\n\n&&\n\nSTRSTARTS(STR(?ortholog_uniprot_uri),\"http://purl.uniprot.org/uniprot/”) )}\n\nThe HBB gene is represented with the UniProt URI http://purl.uniprot.org/uniprot/P68871. To retrieve the orthologs of other genes, we can replace this URI with one that corresponds to another gene such as human INS (i.e. http://purl.uniprot.org/uniprot/P01308 ). We can also provide a set of URIs enclosed with parentheses such as follows: VALUES(?gene_uniprot_uri) {(<http://purl.uniprot.org/uniprot/P68871>)(<http://purl.uniprot.org/uniprot/P01308>) }. The sio:SIO_000558 is the « is orthologous to » property, while the obo:RO_0002162 represents the « in taxon » property (for a graphical abstraction, see Figure 4).\n\nWe note here that not all EBI gene entries have an assigned cross-reference to UniProt. For example, “ENSG00000139618” identifies an Ensembl gene for which the UniProt cross-reference is missing from the EBI RDF platform. In this case, the previous SPARQL query can be adapted, by assigning in the VALUES statement of the query, the ?gene variable to the corresponding Ensembl identifier, as depicted in the following code fragment:\n\nPREFIX obo: <http://purl.obolibrary.org/obo/>\n\nPREFIX sio: <http://semanticscience.org/resource/>\n\nPREFIX ensembl: <http://rdf.ebi.ac.uk/resource/ensembl/>\n\nPREFIX ensemblterms: <http://rdf.ebi.ac.uk/terms/ensembl/>\n\nSELECT DISTINCT ?gene ?ortholog_uniprot_uri {\n\nVALUES(?gene){(ensembl:ENSG00000139618)}\n\n?gene sio:SIO_000558 ?ortholog.\n\n?gene obo:RO_0002162 ?taxon.\n\n?ortholog obo:RO_0002162 ?ortholog_taxon.\n\n?ortholog ensemblterms:DEPENDENT ?ortholog_uniprot_uri.\n\n?gene ensemblterms:DEPENDENT ?gene_uniprot_uri.\n\nFILTER(?taxon != ?ortholog_taxon &&\n\nSTRSTARTS(STR(?ortholog_uniprot_uri),\"http://purl.uniprot.org/uniprot/”) )}\n\nThis code fragment illustrates the SPARQL query to retrieve orthologs for the human BRCA2 gene from the Ensembl dataset. The BRCA2 gene is represented with the UniProt URI ensembl:ENSG00000139618 where ensembl is a prefix that replaces http://rdf.ebi.ac.uk/resource/ensembl/. To retrieve the orthologs of other genes, we can replace ensembl:ENSG00000139618 with a URI that corresponds to another gene such as human INS (i.e. ensembl:ENSG00000254647). We can also provide a set of URIs enclosed with parentheses such as follows: VALUES(?gene){(ensembl:ENSG00000139618)(ensembl:ENSG00000254647)}.\n\nb) Retrieving OMA pairwise orthologs\n\nThe following code fragment shows a SPARQL query to retrieve pairwise orthologs of the human HBB gene which are derived from the HOGs in the OMA database. To execute the query, copy and paste it in the OMA SPARQL endpoint webpage: https://sparql.omabrowser.org/lode/sparql.\n\nPREFIX oma: <http://omabrowser.org/ontology/oma#>\n\nPREFIX orth: <http://purl.org/net/orth#>\n\nPREFIX sio: <http://semanticscience.org/resource/>\n\nPREFIX lscr: <http://purl.org/lscr#>\n\nSELECT DISTINCT ?protein1 ?protein2 {\n\nVALUES(?protein1){(<http://purl.uniprot.org/uniprot/P68871>)}\n\n?cluster a orth:OrthologsCluster.\n\n?cluster orth:hasHomologousMember ?node1.\n\n?cluster orth:hasHomologousMember ?node2.\n\n?node1 orth:hasHomologousMember* ?protein_OMA_1.\n\n?node2 orth:hasHomologousMember* ?protein_OMA_2.\n\n?protein_OMA_1 lscr:xrefUniprot ?protein1.\n\n?protein_OMA_2 lscr:xrefUniprot ?protein2.\n\nFILTER(?node1 != ?node2)}\n\nThe HBB gene is represented in this code fragment with the UniProt URI http://purl.uniprot.org/uniprot/P68871. More precisely, in OMA the lscr:xrefUniprot represents the cross reference to UniProt.\n\nc) Retrieving MBGD pairwise orthologs\n\nIn a similar manner to the previous code fragment, the following depicts a SPARQL query to retrieve pairwise orthologs of the human HBB gene which are derived from the HOGs in the MBGD database. To execute the query, copy and paste it in the MBGD SPARQL endpoint webpage: http://mbgd.genome.ad.jp/sparql/.\n\nPREFIX mbgdr: <http://mbgd.genome.ad.jp/rdf/resource/>\n\nPREFIX mbgd: <http://purl.jp/bio/11/mbgd#>\n\nPREFIX orth: <http://purl.org/net/orth#>\n\nSELECT ?protein1 ?protein2\n\nWHERE {\n\nVALUES(?protein1){ (<http://purl.uniprot.org/uniprot/P68871>)}\n\n?cluster a orth:OrthologsCluster.\n\n?cluster orth:hasHomologous ?node1.\n\n?cluster orth:hasHomologous ?node2.\n\n?node1 orth:hasHomologous* ?gene1.\n\n?node2 orth:hasHomologous* ?gene2.\n\n?gene1 mbgd:uniprot ?protein1.\n\n?gene2 mbgd:uniprot ?protein2.\n\nFILTER(?node1 != ?node2)}\n\nThe HBB gene is represented again with the UniProt URI http://purl.uniprot.org/uniprot/P68871. In the case of MBGD, the mbgd:uniprot represents the cross-reference to UniProt.\n\nIn this protocol we illustrate the task of retrieving the non-hierarchical homologous groups of a target gene, such as the human HBB gene. In addition, we restrict the search to a specific taxonomic level, for example, “only at the primates level”. In other words, we depict how to retrieve the homologous groups at a given taxonomic level and including a given gene represented as a UniProt URI. Note that the same query can be executed only providing one of the inputs (i.e. either the taxonomic level or gene). However, it can take longer to return all results or may not even be executed due to runtime constraints at the original databases. The members of a homologous group can be either paralogous or orthologous to one another.\n\na) Retrieving OMA Homologous Groups derived from the HOGs\n\nThe following code fragment can be executed at the OMA SPARQL endpoint webpage at https://sparql.omabrowser.org/lode/sparql.\n\nPREFIX lscr: <http://purl.org/lscr#>\n\nPREFIX orth: <http://purl.org/net/orth#>\n\nSELECT DISTINCT ?cluster ?protein2_OMA_URI ?protein2_uniprot_URI ?tax_name {\n\nVALUES(?protein1_uniprot_URI){(<http://purl.uniprot.org/uniprot/P68871>)}\n\nVALUES(?tax_name){(\"Primates\")}\n\n?cluster a orth:OrthologsCluster.\n\n?cluster orth:hasHomologousMember* ?protein_OMA_1.\n\n?cluster orth:hasHomologousMember* ?protein2_OMA_URI.\n\n?protein_OMA_1 a orth:Protein.\n\n?protein2_OMA_URI a orth:Protein.\n\n?protein_OMA_1 lscr:xrefUniprot ?protein1_uniprot_URI.\n\nOPTIONAL{?protein2_OMA_URI lscr:xrefUniprot ?protein2_uniprot_URI.}\n\n?cluster orth:hasTaxonomicRange ?tax.\n\n?tax orth:taxRange ?tax_name. }\n\nThis code fragment illustrates the SPARQL query to retrieve homologous groups (i.e. clusters) that contains the human HBB gene in the OMA database. The HBB gene is represented with its related UniProt entry (i.e. the UniProt URI http://purl.uniprot.org/uniprot/P68871). To retrieve the clusters that have other genes, we can replace this URI with one that corresponds to another gene such as human INS (i.e. http://purl.uniprot.org/uniprot/P01308). We can also provide a set of URIs enclosed with parentheses such as follows:\n\nVALUES(?protein1_uniprot_URI) {(<http://purl.uniprot.org/uniprot/P68871>) (<http://purl.uniprot.org/uniprot/P01308>)}. Similarly, we can change the taxonomic level of reference as follows: VALUES(?tax_name) {(\"Hominoidea\")}.\n\nb) MBGD Homologous Groups derived from the HOGs\n\nThe HOGs in MBGD do not provide explicit taxonomic levels at the root level of a HOG. However, the taxon NCBI identifiers of subHOGs (i.e. sublevels) can be extracted in some cases from the cluster URI. Since this requires more advanced knowledge of SPARQL (in particular, for parsing the cluster URIs), we only make it available as part of the Extended data.\n\nc) OrthoDB Homologous Groups\n\nThe following code can be executed at the OrthoDB SPARQL endpoint webpage at https://sparql.orthodb.org.\n\nPREFIX orthodb: <http://purl.orthodb.org/>\n\nPREFIX up: <http://purl.uniprot.org/core/>\n\nSELECT DISTINCT ?groups ?species_name ?protein1_uniprot ?gene1 ?taxLevel_uniprot ?taxLevel WHERE {\n\nVALUES ?protein2_uniprot {<http://purl.uniprot.org/uniprot/P68871>}\n\nVALUES ?taxLevel {\"Primates\"}\n\n?gene2 a orthodb:Gene.\n\n?gene2 orthodb:memberOf ?groups.\n\n?gene1 a orthodb:Gene.\n\n?gene1 orthodb:memberOf ?groups.\n\n?gene1 up:organism ?organism.\n\n?organism a ?taxon.\n\n?taxon up:scientificName ?species_name.\n\n?groups orthodb:ogBuiltAt ?taxLevel_uniprot.\n\n?taxLevel_uniprot up:scientificName ?taxLevel.\n\n?gene2 orthodb:xref ?xref2.\n\n?xref2 orthodb:xrefResource ?protein2_uniprot.\n\n?protein2_uniprot a orthodb:Uniprot.\n\n?gene1 orthodb:xref ?xref.\n\n?xref a orthodb:Xref.\n\nOPTIONAL{\n\n?xref orthodb:xrefResource ?protein1_uniprot.\n\n?protein1_uniprot a orthodb:Uniprot.}\n\n} ORDER BY ?groups, ?taxLevel\n\nThis SPARQL query will retrieve flat homologous groups (i.e. clusters) that contains the human HBB gene in OrthoDB. The HBB gene is represented with its related UniProt entry (i.e. the UniProt URI http://purl.uniprot.org/uniprot/P68871).\n\nIn this protocol we show how to retrieve the HOGs containing a target gene, such as the human HBB gene, in the three orthology databases OMA, MBGD and OrthoDB. The Ensembl dataset in the EBI RDF platform is not considered because it does not provide HOG information.\n\na) Retrieving HOGs from OMA\n\nThe following code fragment can be executed at the OMA SPARQL endpoint webpage at https://sparql.omabrowser.org/lode/sparql.\n\nPREFIX obo: <http://purl.obolibrary.org/obo/>\n\nPREFIX orth: <http://purl.org/net/orth#>\n\nPREFIX taxon: <http://purl.uniprot.org/taxonomy/>\n\nPREFIX up: <http://purl.uniprot.org/core/>\n\nPREFIX lscr: <http://purl.org/lscr#>\n\nSELECT DISTINCT ?root_hog ?species_name ?protein1_uniprot (?protein1 as\n\n?protein1_OMA) ?taxLevel {\n\nVALUES ?protein2_uniprot {<http://purl.uniprot.org/uniprot/P68871>}\n\n?root_hog obo:CDAO_0000148 ?hog_cluster. #has_Root\n\n?hog_cluster orth:hasHomologousMember* ?node1.\n\n?node1 a orth:OrthologsCluster.\n\n?node1 orth:hasTaxonomicRange ?level.\n\n?level orth:taxRange ?taxLevel.\n\n?node1 orth:hasHomologousMember* ?protein1.\n\n?hog_cluster orth:hasHomologousMember* ?protein2.\n\n?protein1 a orth:Protein.\n\n?protein1 orth:organism ?organism.\n\n?organism obo:RO_0002162 ?taxon.\n\n?taxon up:scientificName ?species_name.\n\nOPTIONAL{?protein1 lscr:xrefUniprot ?protein1_uniprot}.\n\n?protein2 a orth:Protein.\n\n?protein2 lscr:xrefUniprot ?protein2_uniprot.\n\n} ORDER BY ?taxLevel\n\nThis SPARQL query will retrieve hierarchical orthologous groups that contain the human HBB gene in the OMA database. The HBB gene is represented with its related UniProt entry (i.e. the UniProt URI http://purl.uniprot.org/uniprot/P68871).\n\nb) Retrieving HOGs from MBGD\n\nThe SPARQL query to retrieve HOGs from MBGD is similar to the previous query over OMA and therefore we make it available as Extended data. As a reminder, although both the OMA and MBGD databases rely on different versions of the ORTH ontology, they structure their HOG data similarly.\n\nc) Retrieving HOGs from OrthoDB\n\nThe following code fragment can be executed at the OrthoDB SPARQL endpoint webpage at https://sparql.orthodb.org. Note that the OrthoDB HOGs often do not contain all taxonomic levels between the orthologous cluster at the highest taxonomic level (i.e. the root) and genes (i.e. leaves).\n\nPREFIX orthodb: <http://purl.orthodb.org/>\n\nPREFIX up: <http://purl.uniprot.org/core/>\n\nSELECT DISTINCT ?hog_root ?species_name ?protein1_uniprot ?gene1 ?taxLevel_uniprot ?taxLevel\n\nWHERE {\n\nVALUES ?protein2_uniprot {<http://purl.uniprot.org/uniprot/P68871>}\n\n?gene2 a orthodb:Gene.\n\n?gene2 orthodb:memberOf ?groups.\n\n?gene2 orthodb:memberOf ?hog_root.\n\nFILTER NOT EXISTS {?hog_root orthodb:ancestralOG ?ancestor.}\n\n?groups orthodb:ancestralOG* ?hog_root.\n\n?gene1 a orthodb:Gene.\n\n?gene1 orthodb:memberOf ?groups.\n\n?gene1 up:organism ?organism.\n\n?organism a ?taxon.\n\n?taxon up:scientificName ?species_name.\n\n?groups orthodb:ogBuiltAt ?taxLevel_uniprot.\n\n?taxLevel_uniprot up:scientificName ?taxLevel.\n\n?gene2 orthodb:xref ?xref2.\n\n?xref2 orthodb:xrefResource ?protein2_uniprot.\n\n?protein2_uniprot a orthodb:Uniprot.\n\n?gene1 orthodb:xref ?xref.\n\n?xref orthodb:xrefResource ?protein1_uniprot.\n\n?protein1_uniprot a orthodb:Uniprot.\n\n} ORDER BY ?hog_root, ?taxLevel\n\nThis SPARQL query will retrieve hierarchical orthologous groups that contains the human HBB gene in the OrthoDB database. The HBB gene is represented with its related UniProt entry (i.e. the UniProt URI http://purl.uniprot.org/uniprot/P68871).\n\nIn this protocol, we show how to compare orthology information across multiple databases with SPARQL 1.1. Although the example in the following code fragment is restricted to OMA and MBGD, similar queries over different combinations of the orthology databases mentioned in this article can be derived based on the Code Fragments in Protocols 1, 2 and 3.\n\nFor a given UniProt entry such as the accession number K9Z723, retrieve orthologs that are only in MBGD, but not in OMA. Alternatively, to retrieve only those that appear in both sources, simply remove the \"NOT\" keyword in the FILTER clause below. To execute the query, copy and paste it in the SPARQL endpoint page of OMA: https://sparql.omabrowser.org/lode/sparql.\n\nPREFIX oma: <http://omabrowser.org/ontology/oma#>\n\nPREFIX orth: <http://purl.org/net/orth#>\n\nPREFIX sio: <http://semanticscience.org/resource/>\n\nPREFIX lscr: <http://purl.org/lscr#>\n\nPREFIX mbgd: <http://purl.jp/bio/11/mbgd#>\n\nSELECT ?protein2 ?species WHERE {\n\nSERVICE<http://sparql.nibb.ac.jp/sparql> {\n\nSELECT ?protein2 ?species where {\n\n?cluster_mbgd a orth:OrthologsCluster.\n\n?cluster_mbgd orth:hasHomologous ?node1_mbgd.\n\n?cluster_mbgd orth:hasHomologous ?node2_mbgd.\n\n?node1_mbgd orth:hasHomologous* ?gene1.\n\n?node2_mbgd orth:hasHomologous* ?gene2.\n\n?gene1 mbgd:uniprot <http://purl.uniprot.org/uniprot/K9Z723>.\n\n?gene2 mbgd:uniprot ?protein2.\n\n?gene2 mbgd:organism ?taxon.\n\nOPTIONAL{?taxon mbgd:species ?species.}\n\nFILTER(?node1_mbgd != ?node2_mbgd) } }\n\nFILTER NOT EXISTS { # keep only those that do not exist in OMA\n\n?cluster a orth:OrthologsCluster.\n\n?cluster orth:hasHomologousMember ?node1.\n\n?cluster orth:hasHomologousMember ?node2.\n\n?node1 orth:hasHomologousMember* ?protein_OMA_1.\n\n?node2 orth:hasHomologousMember* ?protein_OMA_2.\n\n?protein_OMA_1 lscr:xrefUniprot <http://purl.uniprot.org/uniprot/K9Z723>.\n\n?protein_OMA_2 lscr:xrefUniprot ?protein2.\n\nFILTER(?node1 != ?node2) }}\n\nThis federated SPARQL query will retrieve pairwise orthologous genes of the Cyanobacterium-aponinum psb27- gene that are found in the MBGD database but are not present in OMA. The psb27 gene is represented with its related UniProt entry, thus the UniProt URI http://purl.uniprot.org/uniprot/K9Z723.\n\nIn the Extended data, we provide additional examples showing how to retrieve the top 10 entries with most orthologs in OMA and MBGD for a given species, e.g. 'Drosophila melanogaster'. These examples illustrate a few more advanced SPARQL features, such as aggregation and ordering by a criterion in order to select the top N results.\n\n\nConclusion\n\nWe provide four protocols that show how to query evolutionary relationships (pairwise orthologs, as well as HOGs) across four major databases available through SPARQL 1.1 endpoints: EBI, OMA, MBGD and OrthoDB. The protocols presented can serve as a useful starting point for readers interested in an introduction to the RDF data models of these sources, as well as the basics of retrieving orthology information through SPARQL queries. Finally, we have shown how aggregations in SPARQL can be used to quickly generate an overview of the data available in each considered database, and how this data can be compared across the sources.\n\nTo sum up, we hope these protocols provide a useful introduction into analysing evolutionary relationships among genes with SPARQL, as well as enriching these analyses by integrating information from external sources, through federated queries. We encourage readers to experiment with the examples presented in this article, which are provided in the accompanying Jupyter notebook, to be directly re-used or integrated into existing research analysis pipelines. As future work, we plan to integrate the queries in this protocol in the BioQuery search interface (Sima et al., 2019) already available for OMA data at http://biosoda.expasy.org/ in order to enable researchers to directly execute or further refine them in a user-friendly environment.\n\n\nData availability\n\nProtocols available from: https://github.com/biosoda/tutorial_orthology/blob/master/Orthology_SPARQL_Notebook.ipynb\n\nArchived protocols as at time of publication: http://doi.org/10.5281/zenodo.3499928\n\nLicense: CC0\n\nZenodo: Protocols to retrieve orthology information with SPARQL, http://doi.org/10.5281/zenodo.3499928 (Sima & Mendes de Farias, 2019).\n\nThis project contains the following extended data:\n\n- Table 1. \"Cheat sheet\" for RDF data available in the four sources considered in this tutorial. (*) GO annotations can be retrieved from the UniProt RDF store through UniProt cross-references.\n\n- Supplementary protocols: Retrieving MBGD Homologous Groups\n\n- Aggregation queries\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).\n\n\nSoftware availability\n\nSPARQL endpoints (all links include further example queries):\n\n- OMA https://sparql.omabrowser.org/lode/sparql\n\n- OrthoDB https://sparql.orthodb.org/\n\n- EBI https://www.ebi.ac.uk/rdf/services/sparql\n\n- for example orthology queries see \"Ensembl\" category\n\nMBGD http://mbgd.genome.ad.jp/sparql/", "appendix": "Footnotes\n\n1 https://www.w3.org/TR/sparql11-query/#propertypaths\n\n\nReferences\n\nAltenhoff AM, Gil M, Gonnet GH, et al.: Inferring hierarchical orthologous groups from orthologous gene pairs. PLoS One. 2013; 8(1): e53786. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAltenhoff AM, Glover NM, Train CM, et al.: The OMA orthology database in 2018: retrieving evolutionary relationships among all domains of life through richer web and programmatic interfaces. Nucleic Acids Res. 2018; 46(D1): 477–485. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrooksbank C, Bergman MT, Apweiler R, et al.: The European Bioinformatics Institute's data resources 2014. Nucleic Acids Res. 2014; 42(Database issue): D18–25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChiba H, Nishide H, Uchiyama I: Construction of an ortholog database using the semantic web technology for integrative analysis of genomic data. PLoS One. 2015; 10(4): e0122802. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nde Farias TM, Chiba H, Fernández-Breis JT: Leveraging logical rules for efficacious representation of large orthology datasets. s.l., Proceedings of the 10th International Semantic Web Applications and Tools for Healthcare and Life Sciences (SWAT4HCLS) Conference. 2017. Reference Source\n\nDuek P, Gateau A, Bairoch A, et al.: Exploring the Uncharacterized Human Proteome Using neXtProt. J Proteome Res. 2018; 17(12): 4211–4226. PubMed Abstract \| Publisher Full Text\n\nFernández-Breis JT, Chiba H, Legaz-García Mdel C, et al.: The Orthology Ontology: development and applications. J Biomed Semantics. 2016; 7(1): 34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFitch WM: Distinguishing homologous from analogous proteins. Syst Zool. 1970; 19(2): 99–113. PubMed Abstract \| Publisher Full Text\n\nGabaldón T, Koonin EV: Functional and evolutionary implications of gene orthology. Nat Rev Genet. 2013; 14(5): 360–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGlover N, Dessimoz C, Ebersberger I, et al.: Advances and Applications in the Quest for Orthologs. Mol Biol Evol. 2019; 36(10): 2157–2164. PubMed Abstract \| Publisher Full Text\n\nIyappan A, Kawalia SB, Raschka T, et al.: NeuroRDF: semantic integration of highly curated data to prioritize biomarker candidates in Alzheimer’s disease. J Biomed Semantics. 2016; 7: 45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJupp S, Malone J, Bolleman J, et al.: The EBI RDF platform: linked open data for the life sciences. Bioinformatics. 2014; 30(9): 1338–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSima AC, De Farias TM, Zbinden E, et al.: Enabling Semantic Queries Across Federated Bioinformatics Databases. Database (to appear). 2019. Publisher Full Text\n\nSima AC, Mendes de Farias T: Protocols to retrieve orthology information with SPARQL (Version v1.0.0-beta). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3499928\n\nSima AC, Stockinger K, de Farias TM, et al.: Semantic integration and enrichment of heterogeneous biological databases. Methods Mol Biol. In: Evolutionary Genomics. s.l.: Springer, 2019; 1910: 655–690. PubMed Abstract \| Publisher Full Text\n\nW3C SPARQL WORKING GROUP, et al.: SPARQL 1.1 overview. W3C recommendation. World Wide Web Consortium, Cambridge, MA, USA. (accessed 24/10/2019), 2013. Reference Source\n\nUchiyama I, Mihara M, Nishide H, et al.: MBGD update 2018: microbial genome database based on hierarchical orthology relations covering closely related and distantly related comparisons. Nucleic Acids Res. 2018; 47(D1): 382–389. Publisher Full Text\n\nWilliams AJ, Harland L, Groth P, et al.: Open PHACTS: semantic interoperability for drug discovery. Drug Discov Today. 2012; 17(21–22): 1188–1198. PubMed Abstract \| Publisher Full Text\n\nZdobnov EM, Tegenfeldt F, Kuznetsov D, et al.: OrthoDB v9.1: cataloging evolutionary and functional annotations for animal, fungal, plant, archaeal, bacterial and viral orthologs. Nucleic Acids Res. 2017; 45(D1): 744–649. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZerbino DR, Achuthan P, Akanni W, et al.: Ensembl 2018. Nucleic Acids Res. 2018; 46(D1): 754–761. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "55910", "date": "27 Nov 2019", "name": "Andra Waagmeester", "expertise": [ "Reviewer Expertise Semantic web", "Bioinformatics", "data modelling", "ontology 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\nThe authors describe how RDF and SPARQL can be instrumental in combining various resources with knowledge on homologs. The authors take the readers by the hand through some well thought examples of SPARQL queries. They have selected four resources. OMA, OrthoDB, EBI and MBGD. For each resource SPARQL queries are given. The paper is well written and a welcome contribution to the field. However, there are some issues:\nThe paper could be improved with a section on applicable ontologies. This maybe as a subsection under materials, where the authors list the resources. Similarly, the used underlying ontologies/controlled vocabularies could be addressed. They are implicitly described in the data model sections, but since this is paper is also presented as a guideline on how to apply SPARQL, it would help the reader to understand how IRIs which are core to the semantic web are selected. Maybe also describe which ontologies in general apply to the narrative of this paper. See Malone et al1.\nOn various places in the paper the authors argue that SPARQL has a steeping learning curve. I disagree, SPARQL is not different from any other query language. I often argue the contrary, i.e. RDF and SPARQL are rather simple to learn. It is nothing more than wrapping your data in triples and select the proper iri's to represent the knowledge. It can't get any simpler. However, identifying the applicable IRIs and finding a balance between creating IRIs and reusing IRIs makes the process difficult.\n\"Software availability\". I would not call a SPARQL endpoint \"software\". I suggests that the mentioned SPARQL endpoints can be downloaded and installed locally. Maybe change to \"online data access\"?\nThe paper would benefit from a software section, though. If possible it would be nice to show some tool integration. Similar to work by Putman T. et.al.2 where linked data (through Wikidata) is rendered in a webapp.\n\"If any results are presented, are all the source data underlying the results available to ensure full reproducibility?\"\nI answered partly, because the SPARQL example queries do not always get results. The SPARQL query ran on the SPARQL endpoint of the EBI did not get results (The query under \"a) . Retrieving EBI pairwise orthologs\"). Would it be possible to add captions and referencable numbers to the queries. The authors suggest to copy and paste the query to the applicable sparql endpoint. This might lead to coding issues, due different keyboard layouts that exist, worldwide. I would recommend using clickable links that lead to either a query (e.g. https://w.wiki/CrW) or its results (e.g. https://w.wiki/CrX).\nTo deal with this (temporarily?) unavailability of the SPARQL endpoint, I would recommend storing the underlying data as rdf in the github repository which also contains the jupyter notebook mentioned. Or at least a subset big enough to demonstrate the results.\nIt is than possible to reproduce the results using for example the following python gist ====== from rdflib import Graph localGraph = Graph() localGraph.parse(\nquery = \"\"\" SELECT * WHERE { ....\n\n....}\n\n\"\"\" localGraph.query(query) etc ======\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5698", "date": "22 Jul 2020", "name": "Tarcísio Mendes", "role": "Author Response", "response": "Dear Andra Waagmeester, We would like to thank you for the insightful comments and suggestions for improvements. We have revised the article in order to take all comments into account. We thus hope that this new version takes into account the remarks and answers the questions raised. In summary, we have made the following changes: We have added all the queries presented in the article to the BioQuery3 interface online, (available at biosoda.expasy.org), Section \"Information on Homologous Gene queries\", making it easier for readers to try them out directly without requiring copy-pasting the SPARQL queries from the text of the article. Furthermore, we have also created clickable links that point users directly to the corresponding query results in the relevant SPARQL endpoint webpage, in the form of purl.org/orthology/q0 to purl.org/orthology/q9, where the ids Q0-Q9 correspond to the queries presented in the “Protocols” section. We have added a subsection \"Applicable Ontologies\", in the \"Materials\" section, explaining the ontologies relevant to the orthology domain, used by the four data sources described in the article (OMA, OrthoDB, MBGD and EBI). We have improved the explanations regarding the choice of relevant IRIs for formulating SPARQL queries targeting the orthology domain, while clarifying the specific challenges of querying orthology data with SPARQL. We have improved the OrthoDB data model figure with small corrections. We have included a subsection about “retrieving OrthoDB pairwise orthologs”. The following paragraphs list the issues identified by you, along with our corresponding answers. Below we provide detailed answers to your comments. The authors describe how RDF and SPARQL can be instrumental in combining various resources with knowledge on homologs. The authors take the readers by the hand through some well thought examples of SPARQL queries. They have selected four resources. OMA, OrthoDB, EBI and MBGD. For each resource SPARQL queries are given. The paper is well written and a welcome contribution to the field. However, there are some issues: The paper could be improved with a section on applicable ontologies. This maybe as a subsection under materials, where the authors list the resources. Similarly, the used underlying ontologies/controlled vocabularies could be addressed. They are implicitly described in the data model sections, but since this is paper is also presented as a guideline on how to apply SPARQL, it would help the reader to understand how IRIs which are core to the semantic web are selected. Maybe also describe which ontologies in general apply to the narrative of this paper. See Malone et al1. Authors: We have added the subsection \"Applicable Ontologies\" in the \"Materials\" section. We explained the ontologies relevant to the orthology domain used by the four data sources described in the article (OMA, OrthoDB, MBGD and EBI). In particular, we described the Orthology Ontology (ORTH), but also several other controlled vocabularies used across the four data sources, such as GO annotations, the NCBI taxonomy etc. On various places in the paper the authors argue that SPARQL has a steeping learning curve. I disagree, SPARQL is not different from any other query language. I often argue the contrary, i.e. RDF and SPARQL are rather simple to learn. It is nothing more than wrapping your data in triples and select the proper iri's to represent the knowledge. It can't get any simpler. However, identifying the applicable IRIs and finding a balance between creating IRIs and reusing IRIs makes the process difficult. Authors: We have rephrased the abstract to better point out the specific challenges of querying evolutionary data with SPARQL, in particular, formulating property paths to traverse the hierarchical orthologous groups (HOGs). We have also added the new subsections \"Applicable Ontologies\" and \"Choosing the relevant target gene identifiers in RDF\" with improved explanations to identify possible relevant IRIs (for example, based on the applied vocabularies) to compose SPARQL queries in the context of the orthology domain. These queries use UniProt accession numbers as a starting point, and use cross-references available in each of the four data sources (OMA, OrthoDB, MBGD and EBI), to retrieve relevant data for target genes of interest. \"Software availability\". I would not call a SPARQL endpoint \"software\". I suggests that the mentioned SPARQL endpoints can be downloaded and installed locally. Maybe change to \"online data access\"? The paper would benefit from a software section, though. If possible it would be nice to show some tool integration. Similar to work by Putman T. et.al.2 where linked data (through Wikidata) is rendered in a webapp. Authors: We have renamed the previous \"Software availability\" section on SPARQL endpoints to “Online Data Access” as suggested. We also added an entirely new section called \"Software and Online Data Access\", where we describe the integration of the queries in the BioQuery interface available online at biosoda.expasy.org. \"If any results are presented, are all the source data underlying the results available to ensure full reproducibility?\"I answered partly, because the SPARQL example queries do not always get results. The SPARQL query ran on the SPARQL endpoint of the EBI did not get results (The query under \"a) . Retrieving EBI pairwise orthologs\"). Would it be possible to add captions and referencable numbers to the queries. The authors suggest to copy and paste the query to the applicable sparql endpoint. This might lead to coding issues, due different keyboard layouts that exist, worldwide. I would recommend using clickable links that lead to either a query (e.g. https://w.wiki/CrW) or its results (e.g. https://w.wiki/CrX). Authors: We have created clickable links accordingly. These links point readers directly to the corresponding query results in the relevant SPARQL endpoint webpage. The links are in the form of purl.org/orthology/q0 to purl.org/orthology/q9 where the suffixes q0-q9 after the namespace “purl.org/orthology/” correspond to the Q0-Q9 queries presented in the “Protocols” section (and are clearly marked in the article as such). To deal with this (temporarily?) unavailability of the SPARQL endpoint, I would recommend storing the underlying data as rdf in the github repository which also contains the jupyter notebook mentioned. Or at least a subset big enough to demonstrate the results. Authors: Given that copying the data fragments to our github repository might lead to several issues due to licensing problems, as well as to data size, we have made available a preview of the results directly in the Jupyter notebook. We have included this explanation in the article. We have also added a clarification regarding the availability of the SPARQL endpoints and the relevant support addresses to contact (the support provided by each database owner) in case of long unavailability." } ] }, { "id": "55913", "date": "12 Jun 2020", "name": "Ikuo Uchiyama", "expertise": [ "Reviewer Expertise Bioinformatics", "specifically on comparative and evolutionary genomics and genome databases." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, the authors provide introductory examples showing how to query hierarchical orthology databases in RDF format using the SPARQL query language. Although these semantic web technologies are promising for integrating various biological resources, they still seem to be utilized by only very limited researchers. In this respect, this kind of introductory paper may be helpful for more biologists to utilize these technologies. However, for this purpose, I think that this manuscript has a serious defect that it is not easily understandable unless the readers have sufficient knowledge and skills of BOTH SPARQL and homology/orthology concepts (I do not think that this article contains any specific new ideas for such experts). Therefore, the authors should add some more explanation to improve the understandability for those who are not very familiar with either of these fields in order to achieve the goal of this article.\nAt the first example in \"Data models\" section (page 4), the authors should add some explanation about how to make a query based on a given RDF graph (with some figure). Such an explanation in addition to the RDF graphs of the actual databases can help understand the subsequent SPARQL codes.\n\nHere, protocols are shown for extracting three types of homology/orthology information: pairwise orthologs, homologous groups, and hierarchical orthologous groups (HOGs), but the exact definitions of the extracted information are not clearly given. Moreover, the specifications of the protocols including their purposes and outcomes are not clearly explained. In fact, all of these protocols retrieve some kinds of pairwise homology/orthology relationships of a given UniProt entry, but the differences of them are not very obvious. Therefore, it is not easy for readers to understand these protocols exactly, and even impossible to check the correctness of the code, without any appropriate explanation.\n\nRelated but somewhat minor comment: homologous group considered here seems to correspond to orthologous group that contains only orthologs and in-paralogs, while I think homologous group can generally also contain out-paralogs.\n\nMost readers are probably not satisfied with just a copy-and-paste of the given examples, but want to modify them for their own purposes, so it is desirable to give some hints on how to make modifications to each query.\n\nSome codes containing double quotation marks did not work well when I did copy and paste them from the article web site, probably because these characters were converted into non-ASCII characters.\n\nIn the last example, the URIs of the same UniProt entry are appeared repeatedly. Can’t you use a common variable to specify the query URI only once?\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "5699", "date": "22 Jul 2020", "name": "Tarcísio Mendes", "role": "Author Response", "response": "Dear Ikuo Uchiyama, We would like to thank you for the insightful comments and suggestions for improvements. We have revised the article in order to take all comments into account. We thus hope that this new version takes into account the remarks and answers the questions raised. In summary, we have made the following changes: We have added all the queries presented in the article to the BioQuery3 interface online, (available at biosoda.expasy.org), Section \"Information on Homologous Gene queries\", making it easier for readers to try them out directly without requiring copy-pasting the SPARQL queries from the text of the article. Furthermore, we have also created clickable links that point users directly to the corresponding query results in the relevant SPARQL endpoint webpage, in the form of purl.org/orthology/q0 to purl.org/orthology/q9, where the ids Q0-Q9 correspond to the queries presented in the “Protocols” section. We have added a subsection \"Applicable Ontologies\", in the \"Materials\" section, explaining the ontologies relevant to the orthology domain, used by the four data sources described in the article (OMA, OrthoDB, MBGD and EBI). We have improved the explanations regarding the choice of relevant IRIs for formulating SPARQL queries targeting the orthology domain, while clarifying the specific challenges of querying orthology data with SPARQL. We have improved the OrthoDB data model figure with small corrections. We have included a subsection about “retrieving OrthoDB pairwise orthologs”. The following paragraphs list the issues identified by you, along with our corresponding answers. Below we provide detailed answers to your comments. In this manuscript, the authors provide introductory examples showing how to query hierarchical orthology databases in RDF format using the SPARQL query language. Although these semantic web technologies are promising for integrating various biological resources, they still seem to be utilized by only very limited researchers. In this respect, this kind of introductory paper may be helpful for more biologists to utilize these technologies. However, for this purpose, I think that this manuscript has a serious defect that it is not easily understandable unless the readers have sufficient knowledge and skills of BOTH SPARQL and homology/orthology concepts (I do not think that this article contains any specific new ideas for such experts). Therefore, the authors should add some more explanation to improve the understandability for those who are not very familiar with either of these fields in order to achieve the goal of this article. Authors: To improve understandability we have added better explanations and citations throughout this article (see further details in the answers below). At the first example in \"Data models\" section (page 4), the authors should add some explanation about how to make a query based on a given RDF graph (with some figure). Such an explanation in addition to the RDF graphs of the actual databases can help understand the subsequent SPARQL codes. Authors: We have added a new figure in the beginning of the “Data Models” section as well as an explanation regarding how to write a basic SPARQL query starting from this (simplified) graph model. We now also refer readers to an introductory book chapter we recently published on the topic. Here, protocols are shown for extracting three types of homology/orthology information: pairwise orthologs, homologous groups, and hierarchical orthologous groups (HOGs), but the exact definitions of the extracted information are not clearly given. Moreover, the specifications of the protocols including their purposes and outcomes are not clearly explained. In fact, all of these protocols retrieve some kinds of pairwise homology/orthology relationships of a given UniProt entry, but the differences of them are not very obvious. Therefore, it is not easy for readers to understand these protocols exactly, and even impossible to check the correctness of the code, without any appropriate explanation. Authors: We agree that the terminology is confusing. In fact, in the meantime, we have published a separate F1000 paper which introduces the various types of orthologs used in OMA (https://f1000research.com/articles/9-27). We now refer to this, and have also added better explanations of the retrieved results for each query type (e.g. pairwise orthologs, homologous groups, and HOGs). To check the correctness of the retrieved results, the reader has to rely on potentially more time consuming alternative solutions provided by each independent database (e.g. RESTful APIs, Python libraries) or perform interactive searches on the omabrowser.org website. Related but somewhat minor comment: homologous group considered here seems to correspond to orthologous group that contains only orthologs and in-paralogs, while I think homologous group can generally also contain out-paralogs. Authors: The reviewer is correct. We have added a sentence of explanation (“However, the kind of homologous groups considered in this tutorial do not contain “out-paralogs”—i.e. paralogs which result from gene duplications which took place prior to the last common ancestor of all species in the databases”). Most readers are probably not satisfied with just a copy-and-paste of the given examples, but want to modify them for their own purposes, so it is desirable to give some hints on how to make modifications to each query. Authors: We have added an introduction on how to formulate a SPARQL query starting from an example data model graph, also pointing to the Ontology Lookup Service for mapping terms to their RDF identifiers (IRIs). We also indicate to the reader how to formulate SPARQL queries starting from the first data model introduced, MBGD, by following the direction of the arrows in the simplified data model figure. Finally, we have also added a dedicated subsection \"Choosing the relevant target gene identifiers in RDF\" explaining how to change the examples provided with the corresponding target genes of interest. Changing the queries should be facilitated by the availability of direct clickable links to the queries (see explanation below). Some codes containing double quotation marks did not work well when I did copy and paste them from the article web site, probably because these characters were converted into non-ASCII characters. Authors: To solve this problem, we have now created clickable links that point users directly to the corresponding query results in the relevant SPARQL endpoint webpage. This avoids potential errors resulting from copy-pasting the formatted text into the relevant SPARQL endpoint webpage where the query is executed or possibly further modified. Furthermore, we have added all the queries presented in this article also to the BioQuery3 interface online, (available at biosoda.expasy.org), in the Section \"Information on Homologous Gene queries\", making it easier for readers to try them out directly without requiring copy-pasting the SPARQL queries from the text of the article. In the last example, the URIs of the same UniProt entry are appeared repeatedly. Can’t you use a common variable to specify the query URI only once? Authors: We have now replaced the repeated entry with a variable ?uniprot_entry. We also updated the Jupyter notebook accordingly." } ] }, { "id": "64153", "date": "29 Jun 2020", "name": "Tatsuya Kushida", "expertise": [ "Reviewer Expertise Bioinformatics", "Database integration", "RDF", "Ontology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 can be useful to leverage orthologous gene knowledge in genome data analysis like gene function annotations, and the inference. The authors attempted to introduce how to retrieve the orthologous genes for several RDF databases such as OMA, MBGD, OrthoDB, and EnsemblRDF, using concrete SPARQL query examples. In particular, I highly evaluate that the authors explain through appropriate examples of the methods to retrieve the information of the complicated orthologous structures. It is expected to facilitate the understanding of the knowledge, and it would make contributions to increase the usage for genome data analysis. Moreover, the authors provided the Python codes' information to extract orthologous genes from the RDF databases and incorporate them into existing analysis pipelines. That is also worth appreciating. It is essential to further appeal the merits to bioinformaticians and experimental researchers in order to disseminate the orthologous RDF data.\nI realized that as stated in the section: Aggregations in SPARQL: Combining data from multiple resources (page 11), I can aggregate the data more efficiently using the SPARQL than traditional procedure (file-based data exchange or full database dumps.) On the other hand, the authors did not sufficiently mention the advantage of the RDF and SPARQL compared with the traditional procedure. I hope that the authors would more strongly emphasize the advantage.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5700", "date": "22 Jul 2020", "name": "Tarcísio Mendes", "role": "Author Response", "response": "Dear Tatsuya Kushida, We would like to thank you for the insightful comments and suggestions for improvements. We have revised the article in order to take all comments into account. We thus hope that this new version takes into account the remarks and answers the questions raised. In summary, we have made the following changes: We have added all the queries presented in the article to the BioQuery3 interface online, (available at biosoda.expasy.org), Section \"Information on Homologous Gene queries\", making it easier for readers to try them out directly without requiring copy-pasting the SPARQL queries from the text of the article. Furthermore, we have also created clickable links that point users directly to the corresponding query results in the relevant SPARQL endpoint webpage, in the form of purl.org/orthology/q0 to purl.org/orthology/q9, where the ids Q0-Q9 correspond to the queries presented in the “Protocols” section. We have added a subsection \"Applicable Ontologies\", in the \"Materials\" section, explaining the ontologies relevant to the orthology domain, used by the four data sources described in the article (OMA, OrthoDB, MBGD and EBI). We have improved the explanations regarding the choice of relevant IRIs for formulating SPARQL queries targeting the orthology domain, while clarifying the specific challenges of querying orthology data with SPARQL. We have improved the OrthoDB data model figure with small corrections. We have included a subsection about “retrieving OrthoDB pairwise orthologs”. The following paragraphs list the issues identified by the reviewers, along with our corresponding answers. Below we provide detailed answers to your comments. It can be useful to leverage orthologous gene knowledge in genome data analysis like gene function annotations, and the inference. The authors attempted to introduce how to retrieve the orthologous genes for several RDF databases such as OMA, MBGD, OrthoDB, and EnsemblRDF, using concrete SPARQL query examples. In particular, I highly evaluate that the authors explain through appropriate examples of the methods to retrieve the information of the complicated orthologous structures. It is expected to facilitate the understanding of the knowledge, and it would make contributions to increase the usage for genome data analysis. Moreover, the authors provided the Python codes' information to extract orthologous genes from the RDF databases and incorporate them into existing analysis pipelines. That is also worth appreciating. It is essential to further appeal the merits to bioinformaticians and experimental researchers in order to disseminate the orthologous RDF data. I realized that as stated in the section: Aggregations in SPARQL: Combining data from multiple resources (page 11), I can aggregate the data more efficiently using the SPARQL than traditional procedure (file-based data exchange or full database dumps.) On the other hand, the authors did not sufficiently mention the advantage of the RDF and SPARQL compared with the traditional procedure. I hope that the authors would more strongly emphasize the advantage. Authors: We now included a better explanation to emphasize this advantage in the subsection “Aggregations in SPARQL: Combining data from multiple resources”." } ] } ]	1	https://f1000research.com/articles/8-1822
https://f1000research.com/articles/9-587/v1	10 Jun 20	{ "type": "Opinion Article", "title": "SARS-CoV-2 and hypokalaemia: evidence and implications", "authors": [ "Holly Mabillard", "John A. Sayer", "Holly Mabillard" ], "abstract": "The global pandemic secondary to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is leading to unprecedented global morbidity and mortality. With a bewildering array of complications, renal involvement in various forms is common, including serum electrolyte derangements. Hypokalaemia secondary to SARS-CoV-2 was common in a reported Chinese cohort. Here we review the emerging evidence on hypokalaemia and SARS-CoV-2 infection, the potential pathophysiological mechanisms based on early clinical and histopathological data and important clinical implications. Mechanisms of hypokalaemia are multifactorial and so the electrolyte disturbance can be difficult to avoid. We provide further support to the theory of renin-angiotensin-aldosterone (RAS) activation, discuss the strengths and weaknesses of implicating RAS involvement and highlight the importance of calculating the transtubular potassium gradient to identify those at risk of hypokalaemia and its complications.", "keywords": [ "Hypokalaemia", "SARS-CoV-2", "ACE-2", "transtubular potassium gradient", "alkalosis" ], "content": "Introduction\n\nThe global pandemic secondary to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is leading to unprecedented global morbidity and mortality. Anecdotal data and clinical observation from patients with the World Health Organization named disease coronavirus disease 2019 (COVID-19) has highlighted a bewildering array of presentations and pathologies. These include respiratory complications (acute hypoxic respiratory failure, severe pneumonia, acute respiratory distress syndrome (ARDS) and pulmonary fibrosis)1, cardiovascular complications (myocardial injury, myocarditis, cardiomyopathy, cardiac tamponade, acute myocardial infarction, heart failure, dysrhythmias and venous thromboembolic events)2, neurological complications (anosmia, myopathy, acute stroke, Guillain-Barre syndrome and encephalopathy)3, acute liver and/or pancreatic injury4, cytokine storm syndrome, septic shock, DIC, diarrhoea, Kawasaki-like disease5and renal complications (acute tubular injury, rhabdomyolysis, proteinuria, secondary focal segmental glomerulosclerosis and possible renin-angiotensin-aldosterone system activation)6.\n\nSARS-CoV-2 may affect the kidneys directly and indirectly by both renal and renin-angiotensin-aldosterone system (RAS) involvement. What is uncertain are the exact pathophysiological mechanisms that play a role in this. Early reports have suggested that hypokalaemia may be seen frequently in patients infected with the SARS-CoV-2 virus. Here we aim to review the evidence and the potential clinical implications of this well recognised but potentially serious electrolyte disturbance in patients with SARS-CoV-2 infection and touch on renal features of the infection discovered so far.\n\nFirstly, it is important to highlight how transmission of the virus occurs in humans. The SARS-CoV-2 spike protein enters host cells via the angiotensin-converting enzyme 2 (ACE2) receptor on the surface of pulmonary type 2 alveolar cells7,8. ACE2 is crucial to counter-regulate RAS and shares approximately 60% homology with ACE. RAS pathway components are co-expressed in most organs and tissues in the body and communicate via both paracrine and autocrine signalling. ACE2 converts angiotensin-II into angiotensin-(1-7), which acts on the Mas receptor expressed on a variety of tissue cell lineages relevant to cardiovascular disease in addition to type 2 alveolar epithelial cells. It lowers blood pressure through vasodilation and promotion of renal sodium and water excretion and it also attenuates inflammation by producing nitric oxide. Meanwhile, ACE converts angiotensin-I to angiotensin-II which has directly opposing effects to signalling mediated by ACE2. Angiotensin-II acts at the type 1 angiotensin receptor (AT1R) to increase blood pressure by the induction of vasoconstriction and renal sodium and water reabsorption. This also creates oxidative stress which promotes inflammation and fibrosis. The balance between these two opposing pathways determines potential tissue injury, predominantly in the heart and kidneys7,8. Not only has the ACE2 receptor found to be used for host cell entry by the SARS-CoV virus, but the affinity for SARS-CoV-2 is 10-20-fold higher9. During host cell entry, proteolytic cleavage of ACE2 by transmembrane serine protease 2 (TMPRSS2) occurs which is thought to suppress ACE2 expression. This in theory would lead to a reduction in angiotensin-(1-7) generation and angiotensin-II levels would increase resulting in oxidative-stress mediated tissue damage and hypertension7,8. This mechanism resulting in lung parenchymal injury has already been demonstrated in mice injected with SARS-CoV-2 virus10.\n\nInvolvement of the RAS pathway has therefore been speculated as a mechanism to lead to more severe COVID-19 disease and patients with severe disease were more likely to have hypertension, chronic kidney disease (CKD), diabetes mellitus and cardiovascular disease than those with milder disease. This is, however not confirmed and is preliminary data that may be the subject of bias with disregard of age as a potential confounding factor8.\n\nHypokalaemia is known to cause muscle weakness, paraesthesia, thirst, muscle cramps and weakness, but hypokalaemia is an important complication of any disease due to it being a potentially life-threatening condition11. In fact, the incidence of ventricular fibrillation has been shown to be fivefold higher in patients with hypokalaemia compared to those with hyperkalaemia12. Furthermore, inadequate management of hypokalaemia has been identified in 24% of hospitalised patients13, reiterating that we need to be even more vigilant with the management of this life-threatening condition during a pandemic where resources are even more scarce.\n\nThe release of a pre-print retrospective Chinese study initially sparked interest into hypokalaemia as a potentially prevalent biochemical disturbance in SARS-CoV-2 infection14. This was released around a similar time to concerns around RAS inhibitors and SARS-CoV-2 where speculation that RAS inhibitors may increase risk of SARS-CoV-2 infection and its severity due to the knowledge that ACE2 is the viral binding site for host cell entry. It has been stressed by multiple organisations globally that RAS inhibitors should be continued unless otherwise medially indicated as the risks of stopping these important drugs have numerous serious complications to the patient. This study claimed that hypokalaemia was prevalent amongst SARS-CoV-2 infected patients, affecting 108 of 175 patients (62%) that had a serum potassium of <3.5 mmol/l and only 10 patients had a serum K >4.0 mmol/l which is the value required for those with myocardial dysfunction. Of the patients, 22% had severe hypokalaemia (serum potassium <3.0 mmol/l). In total, 11% of all patients and 28% of those with severe hypokalaemia displayed metabolic alkalosis (pH >7.45) compared to 4% with normokalaemia suggesting the possibility of RAS activation. The study suggested that the hypokalaemia was not due to gastrointestinal (GI) loss of potassium as only a small proportion of patients had GI symptoms, there was no significant difference between serum potassium in those with or without diarrhoea and significant urinary potassium wasting was shown. A total of 20 patients had a spot urine potassium/creatinine ratio and there were significantly higher urinary potassium/creatinine ratios in those with hypokalaemia compared with normokalaemia. The study reported that the degree of hypokalaemia correlated with severity of SARS-CoV-2 symptoms and they suggested that hypokalaemia can be difficult to correct as seen in two patients because the renal potassium wasting persists until clinical recovery from the virus. This study is relatively small, limited to a Chinese population and did not disclose medications or diuretic use which could be a significant confounding factor here. As diuretics (especially “loop” diuretics) are used to improve oxygenation in patients with ARDS, some of these patients may well have been prescribed them. It was postulated that hypokalaemia in these patients is due to the effects of increased angiotensin-2 resulting from the proteolytic cleavage of ACE2 from virion invasion of the host although this conclusion cannot be made without measuring components of the RAS system in these patients. Further studies including declaration of medications/diuretic use and measurement of components of the RAS pathway are necessary to make conclusions about aetiology of hypokalaemia in SARS-CoV-2 infection although hypokalaemia was clearly a clinical risk in these patients that should be communicated to the medical community.\n\nA study performed in Hong Kong during the 2003 SARS-CoV epidemic compared symptoms and laboratory findings in SARS-CoV RT-PCR swab-positive patients to negative patients15. Hypokalaemia was present in 41.3% of RT-PCR positive patients and was reported as a ‘common laboratory finding’. It was also suggested in this paper that the hypokalaemia was due to the increased effects of angiotensin-2 but, again, the RAS components were not measured in patients. In contrast, the largest SARS-CoV-2 case series so far (1099 patients included) did not demonstrate any major difference in serum potassium between those with mild and those with severe disease16. Serum potassium was mostly reported as normal in this cohort. SARS-CoV was found in the urine in one patient but urine was not routinely tested for the virus in this cohort.\n\nThe theory of RAS activation causing hypokalaemia is popular, however it is recognised that hyperaldosteronism does not always cause hypokalaemia. This is explained by the ‘renal potassium switch’ mechanism (Figure 1), which involves the paradoxical actions of aldosterone on potassium; for example, aldosterone increases sodium reabsorption in states of low circulating volume without significantly altering potassium balance, but it also increases serum potassium excretion in high potassium states without affecting sodium balance. This paradoxical action all depends on the sodium delivery to the aldosterone-sensitive distal nephron (collecting duct) which is generated by the sodium-chloride co-symporter (NCC) in the early distal convoluted tubule. Activation of this ‘switch’ is by phosphorylation of NCC by the WNK/SPAK/OSR1 pathway17,18 and is done by aldosterone in response to low or high serum potassium i.e. NCC is maximally phosphorylated when serum potassium is <2.5 mmol/l and not at all phosphorylated when serum potassium is >5.2 mmol/l19. In states of decreased circulating volume, ENaC activity is increased due to the effects of excess aldosterone (which generates a sufficient intracellular electrochemical gradient to cause active transport of potassium from blood via Na+K+-ATPase which would be passively excreted into the tubular lumen by ROMK and BK channels in the Principal cell). Whether this generates an effect on potassium or not depends on the integrated activity of the entire distal nephron. In a state of low circulating volume, NCC in the early DCT is ‘switched on’ which results in increased reabsorption of sodium and chloride in the early DCT and, along with sodium reabsorption in the PCT, a net increase of sodium reabsorption from the kidney occurs. As a result, sodium delivery to the aldosterone sensitive distal nephron (collecting duct) is decreased which offsets the effects of ENaC and potassium excretion is therefore unchanged. This results in a state of hyperaldosteronism without hypokalaemia.\n\nIn states of hyperaldosteronism and volume depletion the renal potassium switch is ‘active’. Angiotensin-II (ang-II) stimulates Kir4.1/5.1 to hyperpolarise the basolateral membrane potential and lowers intracellular chloride. This action activates WNK1 (With-no-lysine-kinase-1) and WNK4 (With-no-lysine-kinase-4) by phosphorylation which subsequently phosphorylates SPS1-related proline / alanine-rich kinase (SPAK) and oxidative stress-responsive kinase (OSR1) which, again, phosphorylates the sodium chloride co-symporter (NCC). NCC then avidly reabsorbs sodium (and chloride) back into the body in the early part of the distal convoluted tubule (DCT1). This results in reduced tubular sodium delivery to the collecting duct. Subsequently there is less sodium for the Epithelial sodium Channel (ENaC) to reabsorb and its action is offset. ENaC usually reabsorbs sodium and, via an electrochemical gradient made by sodium-potassium-ATPase, potassium is normally excreted into the tubular lumen by ROMK (Renal Outer Medulla Potassium channel) and BK (Big Potassium/Maxi-K channel) and eliminated from the body. When ENaC is less active, as a consequence of less tubular sodium delivery, less potassium is excreted. This results in a state of hyperaldosteronism with a normal serum potassium. In states of hyperaldosteronism and normal cardiovascular volume (in response to high serum potassium) the switch is ‘inactive’. NCC is not phosphorylated which results in more distal sodium delivery to the collecting duct and subsequent ENaC activation resulting in excretion of potassium into the tubular lumen and a state of hypokalaemia17,18. Arrows represent the direction of movement of electrolytes. Red arrows reflect a reduction in movement and green arrows represent an increase in movement. CLC-Kb, chloride voltage-gated channel Kb; Kir4.1/5.1, inwardly rectifying potassium channel 4.1; AT1R, type 1 angiotensin receptor.\n\nThere are two studies so far (one in pre-print form) to look at renal histopathology on post-mortem findings in SARS-CoV-2 patients who died of the disease (9 of the 26 had pre-morbid clinical kidney injury signs)20,21. The first demonstrated prominent proximal tubule injury (observed in all 26 patients) with frank necrosis observed in some specimens and three patients had high creatinine phosphokinase and pigmented casts on light microscopy, probably representing rhabdomyolysis. There was occasional distal tubule and collecting duct swelling with some interstitial oedema and virus particles were seen in seven of the specimens on electron microscopy in proximal tubule epithelial cells and the podocyte, both of which are the only sites of renal ACE2 expression20. The second post-mortem case series in (currently in pre-print) examined six post-mortem renal histopathology samples of patients who died with SARS-CoV-2 infection21. All specimens had histopathological evidence of acute tubular necrosis, two patients had severe lymphocytic infiltration of the tubulointerstitium and SARS-CoV-2 nucleocapsid antigens were observed in renal tubule cells. Transmission electron microscopy was used in samples from two different patients, both of which demonstrated the presence of virions and virus-like particles in renal cells and these cells were markedly swollen with mitochondrial, lysosomal and endoplasmic reticulum expansion. These finding suggest that SARS-CoV-2 directly infects kidneys and specifically infects and damages kidney tubules. Furthermore, strong CD68+ macrophage presence and C5b-9 deposition seen in the tubulointerstitium of all samples (yet absent in the rest of the kidney tissue and very little in the glomeruli and capillaries in two patients) suggest that further tubular damage occurs as a consequence of macrophage recruitment and C5b-9 activation and deposition. It would be logical to suggest that histopathological evidence of damage to tubular cells would result, clinically, as a tubulopathy, of which we know hypokalaemia is a recognised complication and these case series are histopathological evidence to support this suggestion.\n\nFinally, we know that TMPRSS2 primes SARS-CoV-2 to gain cellular entry and TMPRSS2 is only detectable (in low levels) at the S3 section of the proximal convoluted tubule in the kidney22. It is therefore assumed that this is one way in which the kidney is ‘infected’ by the virus and reiterates the probability of direct virus-induced tubular damage. Various case series have demonstrated the virus in urine23,24 and it is unclear as to how it enters the tubular lumen and whether this is via the apical membrane of tubule cells. However, one study did look at the presence of SARS-CoV-2 in bodily fluids over time and did not detect the virus in urine in any of the nine patients studied25. A couple of case reports have demonstrated that the virus may affect the kidney in other ways which may be an alternative explanation for the entry of virus into the urine26,27. Collapsing focal segmental glomerulosclerosis (FSGS) has been described twice in the literature so far in patients of African or African American origin and both of these patients demonstrated severe tubular injury. It was noted that severe acute tubular necrosis (ATN) was observed in the absence of haemodynamic compromise and severe pulmonary involvement suggesting that the tubulopathy was not ischaemic and more likely to be because of either direct viral toxicity or cytokine-mediated damage.\n\n\nDiscussion\n\nThe data from China and evidence from the SARS-CoV case series provide good clinical justification to monitor potassium levels in patients infected with SARS-CoV-2. We do need to see this clinical finding replicated in multi-centres to be able to make definitive conclusions about this clinical sequela. Although hyperkalaemia can occur for many reasons in patients with SARS-CoV-2 infection, the incidence of ventricular fibrillation has been shown to be five-fold higher in patients with hypokalaemia compared to those with hyperkalaemia12.\n\nBased on evidence of significant urine potassium wasting which can be prolonged, we suggest checking the transtubular potassium gradient (TTKG) in patients with SARS-CoV-2 infection. This would help identify those at risk of severe or prolonged hypokalaemia and prompt pre-emptive cautious electrolyte monitoring and replacement which should help reduce the risk of hypokalaemic complications which can be fatal. The TTKG is easily measured and only requires measurements of serum and urine osmolality and potassium (urine potassium/serum potassium)/(urine osmolality/ serum osmolality). The estimation is relatively accurate providing the urine is not dilute and that urine sodium concentration is >25 mmol/l so that distal sodium delivery is not limiting28.\n\nMeasuring components of the renin-angiotensin-aldosterone pathway would help us to identify the involvement of the SARS-CoV-2 virus; however, measuring these components in the acute setting has its limitations. The RAS system is heavily influenced by a multitude of factors. Many of the patients with SARS-CoV-2 infection are critically unwell and often present to hospital in states of low cardiovascular volume. It has been recommended that patients with ARDS also be kept in a relative state of volume depletion as to not worsen pulmonary interstitial oedema and thus oxygenation. Both of these factors would typically result in RAS activation and could be very misleading when determining virus-related RAS pathway involvement. Some studies have shown that low urine pH (causing acidic urine) can be a predictor of RAS activation29 so future studies determining RAS system involvement of SARS-CoV-2 infection should, as well as TTKG, also consider urine pH as an early marker of RAS system activation.\n\nPotassium levels <3.0 mmol/l can be arrhythmogenic and specifically can cause QTc interval prolongation, torsade de pointes, ventricular fibrillation and sudden cardiac death30. This is particularly relevant for a couple of reasons. First, many of the drugs currently undergoing clinical trials in SARS-CoV-2 patients prolong the QTc interval (hydroxychloroquine31, azithromycin31, favipiravir, lopinavir/ritonavir and fingolimod: NCT04280588) and some drugs also risk hypokalaemia which may worsen pre-existing electrolyte disturbance (e.g. methylprednisolone: NCT04273321, NCT04263402; thalidomide: NCT04273529, NCT04273581; and bevacizumab: NCT04275414). Second, cardiac involvement in SARS-CoV-2 is high (44.4% of infected patients admitted to ICU experienced an arrhythmia). Induction of arrhythmias can be due to multi-factorial aetiologies of cardiac injury in SARS-CoV-2 patients such as hypoxia-mediated, direct tissue damage, cytokine-storm syndrome, worsening coronary perfusion and the direct effects of medications32. Both of these factors really emphasise the importance of maintaining normokalaemia in these patients to reduce morbidity and mortality.\n\nIn addition to the arrhythmogenic effects of both the SARS-CoV-2 cardiac sequelae and various clinical trial drugs, many patients are being given diuretics to improve the oxygenation in ARDS, which also risks hypokalaemic complications. If diuretics are to be used, it would be wise to consider potassium-sparing agents in hypokalaemic patients to reduce the cardiac complications of worsening hypokalaemia that can occur with those that are not potassium sparing.\n\nIt would be logical that with clear histopathological data suggesting tubular injury, hypokalaemia is to be expected in patients infected with SARS-CoV-233. Tubular injury seems to be multifactorial in these patients; ATN from SIRS/Cytokine Storm Syndrome, peritubular congestion, virion invasion, drug toxicity and pigment-cast nephropathy from rhabdomyolysis and therefore difficult to avoid.\n\nThe data on outcomes in cardiac arrest for SARS-CoV-2 patients is unfortunately poor. Chinese data suggests that in a series of 136 patients who underwent resuscitation efforts, return of spontaneous circulation was only achieved in 18 (13.2%) and only four patients were alive at 30 days34. The strong association of hypokalaemia with arrhythmia and sudden cardiac death reiterates the importance of vigilance for detection and treatment of this electrolyte disturbance during and after admission given such poor outcomes when spontaneous circulation is lost.\n\nTo truly know the extent of involvement of the RAS pathway as a potential cause of hypokalaemia in SARS-CoV-2 patients, further studies require measurement of all components of the RAS pathway. Until we know this, we cannot make definite conclusions about the mechanisms of hypokalaemia in these patients outside of the histopathological evidence of tubulopathy published, of which multiple aetiological factors are likely.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nChen JY, Qiao K, Liu F, et al.: Lung transplantation as therapeutic option in acute respiratory distress syndrome for COVID-19-related pulmonary fibrosis. Chin Med J (Engl). 2020. PubMed Abstract \| Publisher Full Text\n\nInciardi RM, Lupi L, Zaccone G, et al.: Cardiac involvement in a patient with coronavirus disease 2019 (COVID-19). JAMA Cardiol. 2020. PubMed Abstract \| Publisher Full Text\n\nMao L, Jin H, Wang M, et al.: Neurologic manifestations of hospitalized patients with coronavirus disease 2019 in Wuhan, China. JAMA Neurol. 2020; e201127. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang F, Wang H, Fan J, et al.: Pancreatic injury patterns in patients with COVID-19 pneumonia. Gastroenterology. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRiphagan S, Gomez X, Gonzalez-Martinez C, et al.: Hyperinflammatory shock in children during COVID-19 pandemic. Lancet. 2020; 395(10237): 1607–1608. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRonco C, Reis T: Kidney involvement in COVID-19 and rationale for extracorporeal therapies. Nature Rev Nephrol. 2020; 16(6): 308–310. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSparks M, South A, Welling P, et al.: Sound Science before Quick Judgement Regarding RAS Blockade in COVID-19. Clin J Am Soc Nephrol. 2020; 15(5): 714–716. PubMed Abstract \| Publisher Full Text\n\nSouth AM, Tomlinson L, Edmonson D, et al.: Controversies of renin–angiotensin system inhibition during the COVID-19 pandemic. Nat Rev Nephrol. 2020; 16(6): 305–307. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWrapp D, Wang N, Corbett KS, et al.: Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 2020; 367(6483): 1260–1263. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKuba K, Imai Y, Rao S, et al.: A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus- induced lung injury. Nat Med. 2005; 11(8): 875–879. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCrop MJ, Hoorn EJ, Lindemas J, et al.: Hypokalemia and subsequent hyperkalemia in hospitalized patients. Nephrol Dial Transplant. 2007; 22(12): 3471–3477. PubMed Abstract \| Publisher Full Text\n\nClausen TG, Brocks K, Ibsen H: Hypokalemia and Ventricular Arrhythmias in Acute Myocardial Infarction. Acta Med Scand. 1988; 224(6): 531–537. PubMed Abstract \| Publisher Full Text\n\nKjeldsen K: Hypokalemia and sudden cardiac death. Exp Clin Cardiol. 2010; 15(4): e96–e99. PubMed Abstract \| Free Full Text\n\nChen D, Xiaokun L, Qifa S, et al.: Hypokalemia and Clinical Implications in Patients with Coronavirus Disease 2019 (COVID-19). MedRxiv. 2020; Publisher Full Text\n\nTsang OTY, Tsang TN, Choi KW, et al.: Coronavirus-positive nasopharyngeal aspirate as predictor for severe acute respiratory syndrome mortality. Emerg Infect Dis. 2003; 9(11): 1381–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuan WJ, Ni ZY, Hu Y, et al.: Clinical Characteristics of Coronavirus Disease 2019 in China. N Engl J Med. 2020; 382(18): 1708–1720. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKamel SK, Schreiber M, Halperin ML: Renal potassium physiology: integration of the renal response to dietary potassium depletion. Kidney Int. 2018; 93(1): 41–53. PubMed Abstract \| Publisher Full Text\n\nMabillard H, Sayer JA: The Molecular Genetics of Gordon Syndrome. Genes. 2019; 10(12): 986. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWelling PA: Roles and Regulation of Renal K Channels. Annu Rev Physiol. 2016; 78: 415–435. PubMed Abstract \| Publisher Full Text\n\nSu H, Yang M, Wan C, et al.: Renal histopathological analysis of 26 postmortem findings of patients with COVID-19 in China. Kidney Int. 2020; S0085-2538(20)30369-0. In press. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDiao B, Wang C, Feng Z, et al.: Human Kidney is a Target for Novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection. MedRxiv preprint. 2020. Publisher Full Text\n\nHoffman M, Schroeder S, Kleine-Weber H, et al.: SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell. 2020; 181(2): 271–280. Publisher Full Text\n\nPeng L, Liu J, Xu W, et al.: 2019 Novel Coronavirus can be detected in urine, blood, anal swabs and oropharyngeal swabs samples. MedRxiv preprint. 2020. Publisher Full Text\n\nWang L, Li X, Chen H, et al.: SARS-CoV-2 infection does not significantly cause acute renal injury: an analysis of 116 hospitalized patients with COVID-19 in a single hospital, Wuhan, China. medRxiv preprint. 2020. Publisher Full Text\n\nWölfel R, Corman VM, Guggemos W, et al.: Virological assessment of hospitalized patients with COVID-2019. Nature. 2020; 581(7809): 465–469. PubMed Abstract \| Publisher Full Text\n\nKissling S, Rotman S, Gerber C, et al.: Collapsing glomerulopathy in a COVID-19 patient. Kidney Int. 2020; S0085-2538(20)30395-1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGaillard F, Ismael S, Sannier A, et al.: Tubuloreticular inclusions in COVID-19-related collapsing glomerulopathy. Kidney Int. 2020; S0085-2538(20)30499-3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nElisaf M, Rizos E, Siamopoulos K: Potassium excretion indices in the diagnostic approach to hypokalaemia. QJM. 2000; 93(5): 318–319. PubMed Abstract \| Publisher Full Text\n\nOgawa S, Nako K, Okamura M, et al.: Lower urinary pH is useful for predicting renovascular disorder onset in patients with diabetes. BMJ Open Diabetes Res Care. 2015; 3(1): e000097.PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWidimsky P: Hypokalaemia and the heart. E-journal of Cardiology Practice. 2008; 7(9). Reference Source\n\nGautret P, Lagier JC, Parola P, et al.: Hydroxychloroquine and azithromycin as a treatment of COVID-19: Results of an open-label non-randomized clinical trial. Int J Antimicrob Agents. 2020; 105949. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZheng YY, Ma YT, Zhang JY, et al.: COVID-19 and the cardiovascular system. Nat Rev Cardiol. 2020; 17: 259–260.Publisher Full Text\n\nSebastian A, McSherry E, Morris RC Jr: Renal Potassium Wasting in Renal Tubular Acidosis (RTA): Its Occurrence in Types 1 and 2 RTA Despite Sustained Correction of Systemic Acidosis. J Clin Invest. 1971; 50(3): 667–78. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShao F, Xu S, Ma X, et al.: In-hospital cardiac arrest outcomes among patients with COVID-19 pneumonia in Wuhan, China. Resuscitation. 2020. 151: 18–23. Publisher Full Text" }	[ { "id": "64575", "date": "26 Jun 2020", "name": "Paul Welling", "expertise": [ "Reviewer Expertise I am an expert on molecular underpinnings of potassium balance and electrolyte disorders. I coined the \"potassium switch\"" ], "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\nAs pointed out by this review, the evidence for hypokalemia in COVID-19 is weak, promulgated by a single unpublished article that was not corroborated by larger series studies. My chief concern about the review, is that it amplifies the myth. Furthermore, the frequent use of loop diuretics in ARDS, which cause hypokalemia, makes a causal connection between the development of hypokalemia and COVID-19 precarious.\n\nAt the very least, the title should be changed to minimize the connection between hypokalemia and COVID-19.\n\nParagraph 1 is somewhat misleading. Although COVID-19 does have an array of different sequela, acute respiratory distress syndrome (ARDS) seems to be the most common serve form of the disease. The first paragraph should provide an incidence of this and the other sequela. Reference to large studies from China, Europe and North American should be cited.\n\nParagraph 2 The statement “SARS-CoV-2 may affect the kidneys directly and indirectly by both renal and renin-angiotensin-aldosterone system (RAS) involvement,” has no factual basis. There are a few small studies that demonstrate that SARS-CoV-2 can infect the kidney but it is presently unclear if this is a key mechanism of AKI in COVID19.\n\nParagraph 3 Provide a reference to Mas receptor on type 2 alveolar epithelial cells as stated. Paragraph 3 last sentence. This is evidence for SARS-COV, not SARS-COV2\n\nProvide a reference to “hyperaldosteronism does not always cause hypokalaemia.” Although “Switch” activation has been a popular explanation for this, it has never been proven. Perhaps, the authors could cite clinical studies, demonstrating the development of severe hypokalemia in normokalemic PA patients following thiazide administration as evidence.\n\nThe limitations of TTKG should be articulated.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-587
https://f1000research.com/articles/9-758/v1	22 Jul 20	{ "type": "Research Article", "title": "Mycobiome analysis in fungal infected formalin-fixed and paraffin-embedded tissues for identification of pathogenic fungi: a pilot study", "authors": [ "Taebum Lee", "Hee Young Na", "Sun-ju Byeon", "Kyoung-Mee Kim", "Hey Seung Lee", "Sung-Hye Park", "Ji-Young Choe", "Kyoung Chan Choi", "Taebum Lee", "Hee Young Na", "Kyoung-Mee Kim", "Hey Seung Lee", "Sung-Hye Park", "Ji-Young Choe", "Kyoung Chan Choi" ], "abstract": "Background: Fungal organisms are frequently observed in surgical pathological diagnosis. In order to more accurately identify fungi in formalin-fixed and paraffin-embedded (FFPE) tissues, it is necessary to use genomic information. The purpose of our pilot study is to identify the factors to be considered for the identification of pathogenic fungi using mycobiome analysis in FFPE tissues. Methods: We selected 49 cases in five hospitals. In each case, FFPE tissue was cut into 50 µm and DNA was extracted. Multiplex PCR with four primers (ITS1, ITS2, ITS3 and ITS4) was performed. Multiplex sequencing was performed using a MinION device according to the manufacturer’s protocol. Sequences of each case were searched using BLASTN with an ITS database from NCBI RefSeq Targeted Loci Project with default parameters. Results: A total of 2,526 DNA sequences were sequenced. We were able to identify 342 fungal sequences in 24 (49.0%, 24/49) cases. The median number of detected fungal sequences per case was 3 (1Q: 1 and 3Q: 14.25). Of the fungal DNA sequences, 215 (62.87%) contained the entire region of ITS1 or ITS2. The remaining 127 fungal DNA sequences were identified as fungi using a partial sequence of ITS1, ITS2, 5.8S, LSU or SSU. Conclusion: In conclusion, we have identified the possibility of finding pathogenic fungi through mycobiome analysis in fungal infected FFPE tissues using nanopore sequencing. However, we have also found several limitations to be solved for further studies. If we develop a method to characterize pathogenic fungi in FFPE tissues in a follow-up study, we think it will help patients to use appropriate antifungal agents.", "keywords": [ "Nanopores", "Fungi", "Genomics", "Mycobiomes" ], "content": "Introduction\n\nFungal organisms are frequently observed in surgical pathological diagnosis. Recent developments in technology have enabled the identification of fungi using a variety of methods1. These novel methods are based on analyzing fungal genomes or proteomes using fresh tissues2,3. However, these methods are difficult to apply in formalin-fixed and paraffin-embedded (FFPE) tissues used in surgical pathology. Sanger sequencing and immunohistochemistry staining have been used in FFPE tissue, but most medical institutions only identify fungi based on morphological findings4–9. The identification of fungi only by morphological findings can lead to inadequate treatment for patients due to misdiagnosis, which can often result in fatal consequences10. As different antifungal agents are preferentially used at the initial infection stage depending on the fungus, it is necessary to identify the exact fungi present through testing methods in addition to the morphological findings11.\n\nIn order to more accurately identify fungi in FFPE tissues, it is essential to use genomic information. DNA markers that could be used to identify fungi include the internal transcribed spacer (ITS) region, small subunit (nrSSU-18S), large subunit (nrLSU-26S or 28S), elongation factor 1-alpha (EF1α), and the largest (RPB1) and second largest (RPB2) subunits of RNA polymerase1,2. Among these markers, ITS could be relatively easily and effectively used for fungal identification, and the database of the ITS regions of fungi is available12–14. In general, sequencing equipment is classified into first- (Sanger sequencing), second- (massively parallel sequencing), and third-generation (real-time and single molecule sequencing) equipment according to key analytical methods15. Third-generation sequencing technology is characterized by direct sequencing of nucleotides without PCR amplification. Oxford Nanopore Technologies (ONT) introduced several sequencing devices using nanopore sequencing technology, which measures the change in current that occurs when a nucleotide sequence passes through a narrow channel16. There is an advantage in identifying each DNA sequence without PCR amplification, and it is expected that the DNA can be effectively detected in spite of DNA degradation during FFPE tissue preparation and storage.\n\nExtracting only fungal DNA from the fungal infection site of FFPE tissue is very difficult. Therefore, we decided to use the mycobiome analysis method. However, no studies have attempted to analyze the mycobiome in FFPE tissue. Therefore, this study was conducted as a pilot study on the development of a method for finding pathogenic fungi using mycobiome analysis in fungal infected FFPE tissues.\n\n\nMethods\n\nThe cases were extracted from the pathological examination reports of five medical institutions. We first extracted pathology reports that mentioned the presence of fungi. We then selected typical cases for use as positive controls, cases reported to be difficult to differentiate (briefly, fungi with branched-hyphae, such as Aspergillus species and Mucor species, are often difficult to distinguish morphologically), and cases with additional information related to fungal identification on the pathology report (i.e., identification using culture or sequencing). Through this process, we finally selected 49 cases (case 3-02 and 5–10 were able to confirm the fungal identification results using sequencing). Cases included 21 lung, eight paranasal sinus, seven gastrointestinal tract, five orbit, two skin and mouth, one adrenal gland, one bone, one gum and one liver samples. The average storage period of FFPE tissue was 4.4 years. This study was exempted from obtaining informed consent by the Institutional Review Board of Hallym University Dongtan Sacred Heart Hospital (NON2018-005).\n\nIn each case, FFPE tissue was cut into 50 µm (5 µm × 10) sections and collected in a 2.0 ml conical tube. DNA was extracted using the ReliaPrep FFPE gDNA Miniprep System (Catalogue number: A2351, Promega, Madison, Wisconsin, USA) according to the manufacturer's protocol. The DNA extracted from all cases and 10µl DNA was loaded onto 1% agarose gel (Certified Molecular Biology Agarose, #1613101, Bio-Rad Laboratories, Hercules, California, USA, Dyne LoadingSTAR with 100bp DNA Ladder, #A751, Dynebio, Seongnam, Korea, Mupid-One, #AD160, Takara Bio, California, USA) with 100V for 30 minutes running to confirm the presence of DNA. Four universal primers (ITS1, 5'-TCCGTAGGTGAACCTGCGG-3'; ITS2, 5'-GCTGCGTTCTTCATCGATGC-3'; ITS3, 5'-GCATCGATGAAGAACGCAGC-3'; and ITS4, 5'-TCCTCCGCTTATTGATATGC-3') were ordered from Macrogen (Seoul, Korea) with polyacrylamide gel electrophoresis purification17. Multiplex PCR with four primers using SimpliAmp Thermal Cycler (ThermoFisher Scientific, Waltham, Massachusetts, USA) and Taq polymerase (New England Biolabs, M0287S, Ipswich, Massachusetts, USA) was performed as follows: initial denaturation at 95°C for 5 min, followed by 35 cycles of 94°C for 30 s, 52°C for 30 s, and 72°C for 1 min with a final extension step of 72°C for 8 min and cooling at 4°C1. Sequencing was performed using the FLO-MIN106 flow cell (ONT, Oxford Science Park, UK), ligation sequencing kit 1D (SQK-LSK109, ONT) and PCR barcoding expansion 1–12 (EXP-PBC001, ONT) with a MinION (ONT) device in accordance with the manufacturer’s protocol (protocol version: PBGE12_9066_v109_revC_23May2018). Multiplex sequencing was performed for 10–12 cases at a time (83–100 ng DNA per sample). The base calling was carried out according to ONT’s recommendations without parameter changes using MinION Software Release 18.12.6.\n\nWe used BLAST+ 2.10.0 on a local PC (Ubuntu 19.10) for analysis18. In brief, we downloaded the ITS database file from NCBI RefSeq Targeted Loci Project (accession number: PRJNA177353, last modified Mar 3, 2020) and then converted it using “makeblastdb” of BLAST+ 2.10.0. This database file contained the full or partial ITS sequence for 11,133 genera of fungi. The RU7 genomic reference sequences of Pneumocystis jirovecii (accession number: GCF_001477535.1), which are not found in this database, were downloaded and compared. The sequence of Actinomyces israelii, a bacterium that looks morphologically similar to that of a filamentous fungus, was downloaded (Actinomyces israelii DSM 43320, whole genome shotgun sequencing project; NZ_JONS00000000.1) and compared. We confirmed the BLASTN search using GRCh38 (accession number: GCF_000001405.26) to determine if the detected DNA corresponds to the human genome.\n\nWe converted FASTQ files to FASTA format using “seqtk” on Github. Sequences of each case were searched using BLASTN using the ITS database file without default parameter modification. When the sequence matched in the ITS database, the result with the highest bit score value was selected. We selected five results in order of highest bit score when five or more fungal DNAs were detected in one case.\n\nWe downloaded the GenBank files of all fungi contained in the database and separated the base sequences of ITS1, 5.8S and ITS2 for each fungus. When fungi were detected, they were classified into three categories (“entire”, “partial”, and “none”) according to the relationship between the detected fungal DNA and ITS1/ITS2. “Entire” means that all ITS1 or ITS2 base information is used for fungal identification, “partial” means that identification is based on a part of ITS1 or ITS2 and its surrounding sequence, and “none” means that base information other than ITS1 or ITS2 is used.\n\nAn earlier version of this article can be found on bioRxiv (https://doi.org/10.1101/2020.04.19.045856).\n\n\nResults\n\nA total of 2,526 DNA sequences were sequenced19. No DNA was sequenced in three cases. Of the remaining 46 cases, the sequenced DNA of 22 cases were not found in the ITS database. We were able to identify 342 fungal sequences in 24 (49.0%, 24/49) cases. The number of DNA sequences identified per case are summarized in Figure 1. The median number of the detected fungal DNA sequences per case was 3 (1Q: 1 and 3Q: 14.25). The clinicopathological information and the number of fungal species detected per case are summarized in Table 1. The remaining cases, in which fungal DNA was not sequenced, are listed in Table 2. Detailed information related to the BLAST program is summarized in Supplementary Table 1 (see Extended data)19. The relationship between the detected fungal DNA and the ITS region is summarized in Table 3 and visualized in Figure 2. Most (215, 62.87%) fungal DNA sequences contained the entire region of ITS1 or ITS2. The remaining 127 fungal DNA sequences were identified as fungi using a partial sequence of ITS1, ITS2, 5.8S, LSU or SSU.\n\nThe mean FFPE block storage period of fungal and no fungal cases was 4.2 years and 4.8 years, respectively (p = 0.410). The DNA concentrations measured by NanoDrop after DNA extraction in the groups with and without fungal DNA detection were 71.2 ng/µl and 104.4 ng/µl, respectively, with no statistical significance (p=0.272). The fungal DNA detection rate was not statistically significant (p=0.376)\n\nRepresentative microscopic images of some cases with inconsistent pathologic diagnosis and mycobiome analysis are summarized in Figure 3 (original images are provided as Underlying data)20. Aspergillus and Candida species were detected by sequencing in case 1-01 (Figure 3A). Compared to the Aspergillus species commonly found in nasal cavities, thinner hyphae were observed, which may have been misleadingly similar to sulfur granules of the pathologically diagnosed Actinomyces. Aspergillus and Acremonium species were detected by sequencing in case 1-03 (Figure 3B). Contrary to case 1-01, it is thought to have been misdiagnosed as Mucor species because its hyphae appeared slightly wider than Aspergillus species. Case 1-08 (Figure 3C) was also misdiagnosed as Actinomyces, in what is thought to be a similar manner to case 1-01. Alternaria, Fusarium and Aspergillus species were detected with numerous sequencing reads. In case 2-08 (Figure 3D), yeast-form fungi were observed in alveolar macrophages using a Gomori methenamine-silver stain and Starmerella cellae was identified by sequencing. Starmerella cellae is a relatively recently identified ovoid to ellipsoidal fungus21. Case 2-09 (Figure 3E) is a fungus found in the pharynx, which shows morphological findings different from those of Candida, Aspergillus, and Mucor. In other words, yeast-form or short branching-type fungal nuclei were found, like in Pneumocystis jiroveci. Cladosporium coloradense was identified by sequencing. Case 3-04 (Figure 3F) is a yeast-form fungus found in subcutaneous tissue and Candida glabrata was identified by numerous sequencing reads.\n\nA) case 1-01, B) case 1-03, C) case 1-08, D) case 2-08, E) case 2-09 and F) case 3-04.\n\nAll three cases diagnosed with Actinomyces were found to have fungi (cases 1-01, 1-02 and 1-08). Case 3-02 was had a pathological diagnosis of Blastomyces dermatitidis by sequencing analysis (summary of sequencing results: 575 nucleotide sequences of U18364.1 were identical without gap opening and 549 nucleotide sequences of EF592163.1 were identical without gap opening). One strand of fungal DNA was identified from the DNA extracted from the FFPE tissue, and the BLASTN analysis showed the highest probability match was Candida africana with a bit score of 468. In this case, the bit score was 154 based on the separate sequence of Blastomyces dermatitidis. Among three cases of pathologically diagnosed cryptococcosis, one case (case 3-10) was found to have a nucleotide sequence of Cryptococcus neoformans with a bit score of 296. In case 4-12, the nucleotide sequence of Candida africana with a bit score of 438 was suggested, with the possibility of Pneumocystis jiroveci with a bit score of 73.1. Four cases of pathologically unidentifiable fungi (cases 2-03, 2-08, 2-09 and 3-04) were found to most likely be Acremonium acutatum, Starmerella cellae, Cladosporium coloradense, and Candida glabrata, respectively.\n\n\nDiscussion\n\nWe performed mycobiome analysis on fungal infected FFPE tissues using nanopore sequencing. The detected fungal DNA occupied approximately one-third of the ITS1 entire region, the ITS2 entire region, and other regions. The advantages of nanopore sequencing compared with Sanger sequencing are as follows. First, unlike Sanger's method, which requires a lot of DNA, nanopore sequencing can be performed with a small amount of DNA (in theory, even with one strand). Second, the nanopore sequencing method can sequence DNA separately, even if the sample contains a variety of lengths of DNA22. Third, nanopore sequencing equipment (i.e., MinION) can be operated at a lower cost (about $1,000) than Sanger sequencing equipment. This low initial cost is a critical factor in the introduction of equipment in small pathology laboratories. Compared to second-generation sequencing equipment, nanopore sequencing has the advantage of sequencing damaged DNA because there is no PCR amplification process in the sequencing process itself. In this study, about one third of the fungal DNA was not an ITS1 entire match or an ITS2 entire match. Therefore, these fungi can be effectively detected using nanopore sequencing.\n\nIt is necessary to find pathogenic fungi among various fungi detected by mycobiome analysis. It may be assumed that dominant fungi are associated with the disease at the site of fungal infection. In cases 1-07 and 1–10, diagnosed as esophageal candidiasis, Candida species comprised 72% and 93% of the fungal DNA, respectively. In such cases, it would be clear to diagnose that the infection is caused by Candida species. In case 1-09, however, only 7% of the fungal DNA was Candida species. Similarly, Rhizopus species were detected in Mucor infections, but as only a small fraction of the fungal DNA. Considering these cases, it is considered desirable to include the process of detecting normal flora in adjacent uninfected sites and selecting pathogenic fungi associated with infection.\n\nWe performed multiplex PCR to increase fungal DNA concentration. However, 2,184 non-fungal DNA sequences accounted for 86.46% of the total DNA. Of the 2,184 DNA sequences not identified as fungi, only 382 (17.50%) were identified in the human genome (GRCh38). Because ribosomal DNA is present in all living organisms, including fungi, humans, and bacteria, it may be difficult to amplify only the fungal ITS region. Nevertheless, we were only able to detect fungal DNA in 49.0% of cases. This detection rate is not high enough to be applied in actual clinical situation and needs improvement. There was no difference in the storage period or DNA quality of the FFPE block between cases where fungal DNA was detected and cases that were not detected. We suggest the following about the causes of low fungal DNA detection rates. The first and most important is a low sequencing output. The third-generation sequencing technique is known to have less sequencing output than the second-generation sequencing equipment. Therefore, increasing the purity of the DNA to be sequenced is important for research. Since we used multiplex sequencing, we were forced to reduce the amount of DNA we analyzed per sample. We believe that the lack of sufficient DNA sequencing in each case was the main cause of the failure to detect fungal DNA. The second is the lack of fungal DNA evaluation methods. Unlike human cells, in which cell viability (closely related to DNA quality) could be predicted by evaluation of a haemotoxylin and eosin stained slide, how to evaluate the viability of fungi in a haemotoxylin and eosin stain is not known. Many studies have shown that inflammation and hypoxia are closely related, and hypoxia induced by inflammation caused by fungal infections may result in damage to fungal DNA and consequently affect the sequencing23. Third, the aforementioned ribosomal DNA is present in almost all living things. We believe this could be overcome by using microdissection, which extracts DNA from fungi as much as possible. Fourth, there is a lack of high-quality databases. Even in the database of NCBI RefSeq Targeted Loci Project, there is only one species (murina) in the Pneumocystis genus. In other words, to diagnose Pneumocystis jiroveci, a separate database should be constructed. In addition, when we analyzed the GenBank file, there were 5,602 ITS1 full sequences and 7,552 ITS2 full sequences in 11,133 genera. Therefore, one well-known fungal ITS DB is not sufficient for identification of fungi, and several DBs must be synthesized to analyze the results. Fifth, it is known that tumor biopsy specimens of 50µm thickness can be used to sufficiently perform next-generation sequencing for diagnostic purposes24. Our study was also performed with a 50µm thickness. However, if thicker tissues (e.g., 100µm thick) were used, the fungal detection rate could be increased.\n\nCandida albicans is the most commonly isolated Candida species in the clinical setting. Of the 162 Candida species, 141 Candida africana (87.04%) and eight Candida albicans (4.94%) sequences were detected. These two Candida species are similar in ITS sequences. Comparing the ITS sequences (ITS1+5.8S+ITS2) of Candida africana (NR_138276.1, 447bp) with Candida albicans (NR_125332.1, 446bp), there is 99.192% identity. There is a 1 bp gap in ITS1 in Candida albicans. Two mismatched nucleotides are located in ITS2, where the DNA base \"C\" in Candida albicans is \"T\" in Candida africana. It is generally known that nanopore sequencing has a slightly higher error rate than second-generation NGS sequencing25. DNA extracted from FFPE tissue is known to have a higher C:T conversion than DNA extracted from fresh frozen tissue26. Because of these, it is difficult to use our method to precisely identify fungi, especially at the species level.\n\nIn summary, we have identified the possibility of finding pathogenic fungi through mycobiome analysis in fungal infected FFPE tissues. However, we have found problems to be solved in further studies, such as increasing sequencing output, increasing fungal DNA concentration, excluding normal flora, and expanding fungal databases. If we develop a method to characterize pathogenic fungi in FFPE tissues in a follow-up study, we think it will help patients to use appropriate antifungal agents.\n\n\nData availability\n\nFigshare: Mycobiome analysis in fungal Infected formalin-fixed and paraffin-embedded tissues for identification of pathogenic fungi: A pilot study. https://doi.org/10.6084/m9.figshare.12616772.v120.\n\nThis project contains the following underlying data:\n\n- Unedited, original microscopy image files\n\nZenodo: byun1114/mycobiome_1: Mycobiome_anlaysis. https://doi.org/10.5281/zenodo.394271119.\n\nThis project contains the following underlying data:\n\n- FASTA.tar (sequencing data in FASTA format)\n\nZenodo: byun1114/mycobiome_1: Mycobiome_anlaysis. https://doi.org/10.5281/zenodo.394271119.\n\nThis project contains the following underlying data:\n\n- SUPPLE_TABLE_1.csv (Supplementary Table 1 containing detailed BLAST results)\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\nRaja HA, Miller AN, Pearce CJ, et al.: Fungal Identification Using Molecular Tools: A Primer for the Natural Products Research Community. J Nat Prod. 2017; 80(3): 756–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAslam S, Tahir A, Aslam MF, et al.: Recent advances in molecular techniques for the identification of phytopathogenic fungi - a mini review. J Plant Interact. 2017; 12(1): 493–504. Publisher Full Text\n\nCassagne C, Normand AC, L'Ollivier C, et al.: Performance of MALDI-TOF MS platforms for fungal identification. Mycoses. 2016; 59(11): 678–90. PubMed Abstract \| Publisher Full Text\n\nMcNeil CJ, Luo RF, Vogel H, et al.: Brain abscess caused by Phaeoacremonium parasiticum in an immunocompromised patient. J Clin Microbiol. 2011; 49(3): 1171–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoncada PA, Budvytiene I, Ho DY, et al.: Utility of DNA sequencing for direct identification of invasive fungi from fresh and formalin-fixed specimens. Am J Clin Pathol. 2013; 140(2): 203–8. PubMed Abstract \| Publisher Full Text\n\nBenamu E, Yu AT, Xie L, et al.: Scedosporium apiospermum infection of the urinary system with a review of treatment options and cases in the literature. Transpl Infect Dis. 2018; 20(1): e12804. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrachuk P, Szymczak WA, Muscarella P, et al.: A Case of Invasive Gastrointestinal Mycotypha Infection in a Patient with Neutropenia. Case Rep Infect Dis. 2018; 2018: 5864175. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGuarner J, Brandt ME: Histopathologic diagnosis of fungal infections in the 21st century. Clin Microbiol Rev. 2011; 24(2): 247–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJung J, Park YS, Sung H, et al.: Using immunohistochemistry to assess the accuracy of histomorphologic diagnosis of aspergillosis and mucormycosis. Clin Infect Dis. 2015; 61(11): 1664–70. PubMed Abstract \| Publisher Full Text\n\nHong KH, Kim JW, Jang SJ, et al.: Liver cirrhosis caused by Exophiala dermatitidis. J Med Microbiol. 2009; 58(Pt 5): 674–7. PubMed Abstract \| Publisher Full Text\n\nPerfect JR: The antifungal pipeline: a reality check. Nat Rev Drug Discov. 2017; 16(9): 603–16. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchoch CL, Seifert KA, Huhndorf S, et al.: Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc Natl Acad Sci U S A. 2012; 109(16): 6241–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchoch CL, Robbertse B, Robert V, et al.: Finding needles in haystacks: linking scientific names, reference specimens and molecular data for Fungi. Database (Oxford). 2014; 2014: bau061. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNilsson RH, Larsson KH, Taylor AFS, et al.: The UNITE database for molecular identification of fungi: handling dark taxa and parallel taxonomic classifications. Nucleic Acids Res. 2019; 47(D1): D259–D64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShendure J, Balasubramanian S, Church GM, et al.: DNA sequencing at 40: past, present and future. Nature. 2017; 550(7676): 345–53. PubMed Abstract \| Publisher Full Text\n\nBranton D, Deamer DW, Marziali A, et al.: The potential and challenges of nanopore sequencing. Nat Biotechnol. 2008; 26(10): 1146–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWhite TJ, Bruns T, Lee S, et al.: Amplification and direct sequencing of fungal ribosomal RNA genes For phylogenetics. 1990. Reference Source\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\nbyun1114: byun1114/mycobiome_1: Mycobiome_anlaysis (Version v1.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3942711\n\nByeon S, Lee T, Na HY, et al.: Mycobiome analysis in fungal Infected formalin-fixed and paraffin-embedded tissues for identification of pathogenic fungi: A pilot study. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12616772.v1\n\nSantos ARO, Leon MP, Barros KO, et al.: Starmerella camargoi f.a., sp. nov., Starmerella ilheusensis f.a., sp. nov., Starmerella litoralis f.a., sp. nov., Starmerella opuntiae f.a., sp. nov., Starmerella roubikii f.a., sp. nov. and Starmerella vitae. f.a., sp. nov., isolated from flowers and bees, and transfer of related Candida species to the genus Starmerella as new combinations. Int J Syst Evol Microbiol. 2018; 68(4): 1333–43. PubMed Abstract \| Publisher Full Text\n\nDe Filippis F, Laiola M, Blaiotta G, et al.: Different Amplicon Targets for Sequencing-Based Studies of Fungal Diversity. Appl Environ Microbiol. 2017; 83(17): e00905–17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEltzschig HK, Carmeliet P: Hypoxia and inflammation. N Engl J Med. 2011; 364(7): 656–65. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCho M, Ahn S, Hong M, et al.: Tissue recommendations for precision cancer therapy using next generation sequencing: a comprehensive single cancer center's experiences. Oncotarget. 2017; 8(26): 42478–86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeirather JL, de Cesare M, Wang Y, et al.: Comprehensive comparison of Pacific Biosciences and Oxford Nanopore Technologies and their applications to transcriptome analysis. F1000Res. 2017; 6: 100. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSpencer DH, Sehn JK, Abel HJ, et al.: Comparison of clinical targeted next-generation sequence data from formalin-fixed and fresh-frozen tissue specimens. J Mol Diagn. 2013; 15(5): 623–33. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "67771", "date": "04 Aug 2020", "name": "Wissam Faour", "expertise": [ "Reviewer Expertise Inflammation", "Cell signaling", "cell biology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors suggested genomic tools to assess fungal infection by manipulating DNA extracted from paraffin-embedded tissue blocks. There is a growing importance of using FFPE tissues further in clinical assessment, therefore, the study continues to contribute to this field. The paper fits within the scope of the journal\nThe paper is well-written in good English.\n\nThe authors need to show the concentrations of the recovered DNA and how they quantify it.\n\nHow the authors controlled for damaged DNA in sequences negative for fungal DNA.\n\nThe study lack proper positive and negative control (can use FFPE known to be free of fungal infection).\n\nMaybe showing a photo of the 1% non-denaturing agarose gel is useful to assess the quality of the isolated DNA.\n\nIs the work 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": "5800", "date": "06 Aug 2020", "name": "Sun-ju Byeon", "role": "Author Response", "response": "Thank you for your kindly comments. Q: The authors need to show the concentrations of the recovered DNA and how they quantify it.A: We measured the concentration of the extracted DNA using Nanodrop. We described using Nanodrop in Page 8, but omitted the description in the method. Q: How the authors controlled for damaged DNA in sequences negative for fungal DNA.A: We have not been able to conduct further experiments on cases where fungal DNA was not found. The research fund we received for this study is not large, and the research fund has been exceeded to purchase MinION devices and reagents needed for sequencing. We discussed in a discussion how to increase the likelihood of detecting fungal DNA because it is not a condition to conduct additional experiments. At the end of this study, we plan to apply for research funds for follow-up studies that can complement the limitations in this study.Q: The study lack proper positive and negative control (can use FFPE known to be free of fungal infection).A: We evaluated the adequacy of the experiment using Candida (labeled #1-11) and Aspergillosis spp. (labeled #1-12) cultured in the Department of Laboratory Medicine as a positive control. We uploaded the FASTA file for the this sample (https://doi.org/10.6084/m9.figshare.12762431.v2). Since ITS1~3 primer can amplify human DNA, a separate negative control was not set. Q: Maybe showing a photo of the 1% non-denaturing agarose gel is useful to assess the quality of the isolated DNA.A: We uploaded an agarose gel image of genomic DNA (https://doi.org/10.6084/m9.figshare.12762422.v1)." } ] }, { "id": "75308", "date": "02 Dec 2020", "name": "Chengqiang Wang", "expertise": [ "Reviewer Expertise Analysis of microbial diversity. Molecular and genetic 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 authors have identified the mycobiome analysis to studying pathogenic fungi in fungal infected FFPE tissues by simple nanopore sequencing. It is a method to identify microbiological compositions in different samples, although the accuracy of this method is not sufficient for diagnosing pathogenic fungi. The experimental treatment and result analysis of this paper are permissible. Overall, the paper fits the scope of the journal. I just present some of my opinions for thinking.\nI think that all ITS1 or ITS2 base information is more accurate for fungal identification, and so, a part of ITS1 or ITS2 sequences might be false positive.\n\nThe method used in this paper is suitable for genuses identification, and so, species identification is not sufficient. For example, “two Candida species are similar in ITS sequences”. I suggest using characteristic genes of species for species identification.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-758
https://f1000research.com/articles/9-256/v1	14 Apr 20	{ "type": "Research Article", "title": "Development and validation of the English version of the Moral Growth Mindset measure", "authors": [ "Hyemin Han", "Kelsie J. Dawson", "YeEun Rachel Choi", "Youn-Jeng Choi", "Andrea L. Glenn", "Kelsie J. Dawson", "YeEun Rachel Choi", "Youn-Jeng Choi", "Andrea L. Glenn" ], "abstract": "Background: Moral Growth Mindset (MGM) is a belief about whether one can become a morally better person through efforts. Prior research showed that MGM is positively associated with promotion of moral motivation among adolescents and young adults. We developed and tested the English version of the MGM measure in this study with data collected from college student participants. Methods: In Study 1, we tested the reliability and validity of the MGM measure with two-wave data (N = 212, Age mean = 24.18 years, SD = 7.82 years). In Study 2, we retested the construct validity of the MGM measure once again and its association with other moral and positive psychological indicators to test its convergent and discriminant validity (N = 275, Age mean = 22.02 years, SD = 6.34 years). Results: We found that the MGM measure was reliable and valid from Study 1. In Study 2, the results indicated that the MGM was well correlated with other moral and positive psychological indicators as expected. Conclusions: We developed and validated the English version of the MGM measure in the present study. The results from studies 1 and 2 supported the reliability and validity of the MGM measure. Given these, we found that the English version of the MGM measure can well measure one’s MGM as we intended.", "keywords": [ "moral growth mindset", "growth mindset", "reliability", "validity", "moral development" ], "content": "Introduction\n\nIn the present study, we aimed to create and validate the English version of the Moral Growth Mindset (MGM) measure, which was originally developed in Korean. Growth mindset refers to the belief that it is possible to improve aspects of one’s life, such as intelligence or personality1. Those with growth mindset generally have higher motivation based on attitudes such as viewing hardships as a chance to work harder rather than an indication of failure, and genuinely wanting to learn instead of being concerned with how others view them2. Since those with growth mindset believe that their skills and abilities can be improved through effort and learning, their motivation is fostered. A study found that an intervention that taught students how to endorse a growth mindset reduced levels of aggression as well as depressive symptoms that resulted from being a victim of bullying3. It suggested that growth mindset might be beneficial for promoting a sense of resilience when faced with social challenges or other difficulties.\n\nMGM refers to growth mindset in the domain of morality. This mindset is related to one’s belief that it is possible to become a morally better person and improve one’s morals through efforts. A study showed that MGM was positively associated with increases in voluntary service engagement among adolescents and young adults4. It suggested that MGM might increase participants’ prosocial behavior due to the belief that participating in this type of behavior will make them morally better among youth. Given this, MGM would be considered as a factor that contributes to moral development.\n\nMGM was previously included as a three-item subscale in a general measure of growth mindset called the Theory Measures5,6. However, because it is important to include four or more items per factor to perform psychometric tests7, the psychometrical qualities of the MGM subscale could not be sufficiently tested in the original studies. In a previous study4, we developed and tested a Korean version of the MGM measure. We evaluated the internal consistency and structure of the measure, but the test-retest consistency and discriminant validity of the measure were not examined. Hence, in the present study, we created an English version of the MGM measure and tested its psychometric properties. In Study 1, we tested the internal and test-retest consistency and validity of the MGM measure and modified the measure to improve the model fit. In Study 2, we examined correlations between the MGM and other moral and positive psychological indicators associated with positive youth development to test the convergent and discriminant validity of the measure.\n\n\nStudy 1\n\nIn Study 1, we translated the MGM measure to English and tested its reliability and validity with two-wave data. We also modified the items to improve the model fit.\n\nTranslation of the MGM measure to English. Based on the Korean version of the MGM measure4 and the Implicit Theory measure1,8, we developed the English version of MGM measure. Although the English version was created based on the Korean version, we did not do direct translation because of cultural differences in concepts and terms related to morals and characters (e.g., 9). Instead, the inventors (HH, KJD, and YJC) of the Korean MGM measure created its English version based on the structure of the Korean version and the wordings in the Implicit Theory measure. The tested measure included six items (e.g., “No matter who you are, you can significantly improve your morals and character”) and answers were anchored to a six-point Likert scale (see Extended data for the full measure10).\n\nAlthough Chiu, Hong, and Dweck11 originally used more nuanced keywords such as “responsible and sincere” as well as “conscientiousness, uprightness, and honesty,” we decided to use the more general terms, “morals and character.” This was due to the concern that such nuanced terms in the original measure may be associated with specific moral foundations and biased towards certain groups of people. In fact, conservatives have been found to score higher on measures of conscientiousness12 whereas liberals have been found to rely primarily on the value of fairness, which is closely related to honesty, when dealing with moral issues (see research on Moral Foundation Theory; e.g., 13). Thus, we used “morals and characters” in order for participants to be able to define morality without bias.\n\nOur measure is in line with the original measure consisting of six items1. In fact, although all of the items were meant to measure whether or not participants endorse a growth mindset and seemingly similar to each other, the wordings varied slightly to include core concepts of growth mindset such as being able to improve regardless of who you are (i.e., “no matter who you are”), the point in time (i.e., “always”), or the degree (i.e., “considerably”). In addition, because we were interested in whether MGM can be differentiated from general growth mindset measured by the original growth mindset measure, we decided to use the same terms and format that were adopted in the original measure.\n\nParticipants. Study 1 was conducted during the 2018 fall semester. Participants were recruited from an undergraduate subject pool. The pool consisted of students who were enrolled in educational psychology classes. Only the students who were enrolled in either of the aforementioned classes and at least 18 years of age were eligible to complete the survey. Participants who were younger than 18 years of age were excluded from the present study. The participants visited the subject pool system, checked the list of active research projects, and selected and signed up for our study. We decided to recruit at least 200 participants since N = 200 has been regarded as the recommended minimum sample size for confirmatory factor analysis (CFA)14.\n\nA total of 212 college students (89.15% females; Age mean = 24.18 years, SD = 7.82 years; 177 Caucasian, 34 African American, 1 Native American, 1 Asian, 1 Pacific Islander, 3 Latinx, 2 multi-ethnic) from the southern USA completed the English MGM measure online via Qualtrics. They were re-invited to complete the same survey again one week later (N = 207 for Wave 2; 89.37% females; Age mean = 24.28 years, SD = 7.88 years).\n\nParticipants received a link to the Qualtrics survey where they completed the MGM measure, followed by a demographics survey. Only the participants who voluntarily signed up for study 1 were provided with the link. When the participants signed up for the study, the subject pool manager provided us with their email addresses, and we sent the participants the survey link via email. We created our Qualtrics survey in a way so that only the participants who answered all survey questions were able to complete the survey and receive a credit for their class. Thus, there was no missing data in the present study.\n\nA consent form was sent out to the students alongside the MGM measure. This form was reviewed by the Institutional Review Board at the University of Alabama (IRB approval numbers for studies 1 and 2: 18-04-1156, 18-10-1633, 18-12-1842), who also approved the studies, and was presented at the beginning of the Qualtrics form. Only students who read the form and agreed to participate in this study were presented with the survey forms.\n\nAnalysis. When examining test-retest reliability, we excluded participants who failed to complete the second survey within two weeks to control for the time gap between the two surveys as an effort to minimize the dispersion of the time gap, which left 168 cases for examining test-retest reliability (Mean time gap between Waves 1 and 2 = 7.78 days, SD = 1.66 days).\n\nFirst, we examined consistency indicators, i.e., Cronbach’s α and test-retest consistency. Second, we performed CFA to examine the internal structure of the measure. We used robust weighted least squares (WLSMV) because it is more suitable for testing Likert-type items in a small sample15. During this process, we checked whether any item should be excluded from the measure to achieve a good model fit. If the measure was modified, we calculated all reliability and validity indicators again. We used R (3.6.1) for statistical analyses. All data files and source codes are available as Underlying data10.\n\nFirst, all consistency indicators indicated that the measure demonstrated at least acceptable reliability (> .7; see Table 1). Second, we performed CFA – the original model with all six items did not show good model fit (see Table 1). Thus, we excluded items 1 and 2 while referring to Han et al. (2018), because in that study we showed the relatively lower factor loadings in the six-item and five-item models respectively. The CFA demonstrated that the four-item model was the best model given excellent model fit indicators (chi-square test p-value > .05, RMSEA and SRMR < .05, TLI and CFI > .95; see Table 2 for the best model16). As shown in Table 1, when we recalculated reliability indicators after exclusion of the items, all indicators remained greater than .7.\n\nIn addition to the low factor loadings, we also decided to remove items 1 and 2 due to the fact that they may have been too vague. For example, item 1 stated “you can’t really do much” and item 2 stated “you can’t improve very much” whereas the other items used words such as “significantly improve,” “always substantially improve,” and “improve…considerably” that conveyed more specific magnitude. Using the less extreme terms in items 1 and 2 may have put the items at risk of inconsistency17 since it would be easier for participants’ opinions to shift regarding whether or not you can change “much.”\n\n\nStudy 2\n\nIn Study 2, we tested correlation between MGM and other moral and positive psychological indicators associated with positive youth development. In addition, we performed CFA for model confirmation.\n\nParticipants. As per Study 1, participants were recruited from the educational psychology and psychology subject pools during the 2019 spring semester. Only the students who were enrolled in the aforementioned classes and at least 18 years of age were eligible to complete the survey. Participants who were younger than 18 years of age were excluded from the present study. Participants in educational psychology classes visited the subject pool system, checked the list of active research projects, and selected and signed up for our study. Participants in psychology classes who intended to sign up for our study visited the SONA system, reviewed the list of active studies, and then selected and signed up for the present study.\n\nWhen participants signed up for the present study, in the cases of educational psychology students, the subject pool manager provided us with their email addresses, and we sent the participants the survey link via email. In the cases of psychology students, they were automatically provided with a link to a Qualtrics survey via the SONA system. They were presented with the MGM measure and other moral and positive psychological measures, all of which were presented in a randomized order, followed by a demographics survey. Similar to Study 1, only the participants who answered all questions were able to complete the survey and receive a credit, so there was no missing data in the present study. For sample size estimation, similar to Study 1, we followed the guidelines for CFA14, so we determined that at least 200 participants were required.\n\nIn total, 275 college students (81.45% females; Age mean = 22.02 years, SD = 6.34 years; 223 Caucasian, 39 African American, 2 Native American, 1 Asian, 1 Pacific Islander, 5 Latinx, 4 multi-ethnic) in the Southern United States of America were recruited. The consent procedure was identical to that in Study 1.\n\nMeasures. MGM measure. We used the four-item MGM measure used in Study 1.\n\nMoral and positive psychological measures for convergent validity check. To test the convergent validity of the MGM measure, we employed moral and positive psychological measures. These include the Implicit Theory Measure1,8, Behavioral Defining Issues Test (bDIT)18,19, Interpersonal Reactivity Index20, Moral Identity Scale21, Propensity to Morally Disengage Scale22, and Claremont Purpose Scale23. Further details regarding these measures, such as brief descriptions and links or citations to full measures, are provided as Extended data10.\n\nAnalysis. First, we performed CFA with the MGM data again to test the internal structure of the MGM measure. Second, we performed correlation analyses to examine how MGM was associated with other moral and positive psychological indicators. According to the previous studies that examined the relationship between growth mindset, positive psychological indicators, and antisocial tendency (e.g., 24–26), we hypothesized that the sizes of correlation coefficients between MGM and other indicators would be between .10 and .30. Third, we tested whether discriminant validity existed between MGM and general growth mindset in order to determine if the MGM measure examines a construct independent from general growth mindset. The examination of discriminant validity was tested with the Fornell-Larcker criterion27.\n\nWe also used R in Study 2. All data files and source codes are available as Underlying data28.\n\nThe results of the reliability check showed that the MGM measure as well as all other measures possessed at least acceptable reliability (> .7; see Table 3). Moreover, CFA supported good internal structure of the MGM measure (see Table 1 and Table 2).\n\nNote. M: mean. SD: standard deviation. r: Pearson correlation coefficient. Cronbach αs are also reported.\n\n† p < .10, * p < .05, p < .01, * p < .001.\n\n1: MGM, 2: general growth mindset, 3: bDIT, 4: IRI EC, 5: IRI PD, 6: IRI PT, 7: IRI FS, 8: moral internalization, 9: moral symbolization, 10: moral disengagement, 11: CPS all, 12: CPS meaningfulness, 13: CPS goal orientation, 14: CPS beyond-the-self dimension.\n\nCorrelation analysis demonstrated a positive association between MGM, general growth mindset, and other moral psychological indicators in general except those relatively less relevant to morality among individuals, such as personal distress, symbolization, and meaningfulness (see Table 3). MGM was marginally correlated with the bDIT and was not significantly correlated with PD and CPS meaning. The correlation between MGM and moral disengagement was significantly negative. We found that the correlation coefficient between MGM and general growth mindset (r = .37) was smaller than the square root of the average variance extracted (AVE=.84), so MGM showed discriminant validity from general growth mindset.\n\n\nDiscussion\n\nWe developed and tested the English version of the MGM measure in this study with data collected from youth participants. In Study 1, we found that the four-item MGM measure possessed good consistency and internal structure. In Study 2, we found that MGM was positively associated with moral and positive psychological indicators as hypothesized. In addition, moral disengagement was negatively correlated with MGM. Furthermore, we found good discriminant validity between the MGM measure and the general growth mindset measure.\n\nOur results from both studies suggest that the English version of the MGM measure can well measure one’s MGM as we intended. In fact, the previous studies that developed and tested measurements for the mindset with diverse domains have shown that the measurements possessed good reliability and validity (e.g., 29,30), so growth mindset can be feasibly measured with self-report measures. Consistent with previous studies about measuring growth mindset in other domains, we were able to show that the MGM can also be appropriately measured by a self-report measure, the MGM measure. Moreover, the results from our correlation analysis are consistent with findings in previous studies that have examined the positive relationship between growth mindset and successful social adjustment and positive youth development in general2,24,28. Hence, our study that tested and validated the MGM measure demonstrated that first, MGM can be well measured by the MGM measure as growth mindset in general was measured by reliable and valid tools in previous studies; and second, MGM is associated with moral and positive youth development as shown in previous growth mindset studies in other domains.\n\nThis English version of the MGM measure will contribute to research in moral development and education. For instance, researchers and educators who are interested in how MGM is associated with moral development may use the MGM measure in their studies. In addition, given that we created the English version of MGM measure, scholars who are using languages other than Korean or English will be able to translate the measure into their languages. By doing so, it would be possible to accumulate large-scale datasets for testing the measure in diverse backgrounds and contexts, and to examine the roles of MGM in moral development in the long term.\n\nHowever, there are limitation in this study that warrant future studies. First, we collected data only from undergraduate students and male students were underrepresented in both studies; such issues may limit the generalizability of our findings. Second, due to the small sample size, we could not conduct CFA for the measures used in Study 2 although they were validated in previous studies. Third, although we used unnuanced terms (e.g., morals and characters), we could not test whether the measure was actually unbiased according to one’s political orientation of endorsed moral foundations; measurement invariance test is needed to examine the point.\n\n\nData availability\n\nOpen Science Framework: Moral Growth Mindset is Associated with Change in Voluntary Service Engagement, https://doi.org/10.17605/OSF.IO/AC6RH10.\n\nThis project contains the following underlying data:\n\n- Data and source code files that support the findings of this study (contained in folder ‘English version MGM’).\n\n- MGM measure in English and information about additional moral and positive psychological measures used in Study 2 (contained in folder ‘English version MGM’, Supplementary materials.docx)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nDweck CS: Self-Theories: Their Role in Motivation, Personality, and Development. Philadephia, PA: Psychology Press. 2000. Reference Source\n\nYeager DS, Dweck CS: Mindsets that promote resilience: When students believe that personal characteristics can be developed. Educ Psychol. 2012; 47(4): 302–14. Publisher Full Text\n\nYeager DS, Trzesniewski KH, Dweck CS: An implicit theories of personality intervention reduces adolescent aggression in response to victimization and exclusion. Child Dev. 2013; 84(3): 970–88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHan H, Choi YJ, Dawson KJ, et al.: Moral growth mindset is associated with change in voluntary service engagement. PLoS One. Lamm C, editor. 2018; 13(8): e0202327. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChiu C, Dweck CS, Tong JY, et al.: Implicit theories and conceptions of morality. J Pers Soc Psychol. 1997; 73(5): 923–40. Publisher Full Text\n\nDweck CS, Chiu C, Hong Y: Implicit theories and their role in judgments and reactions: A word from two perspectives. Psychol Inq. 1995; 6(4): 267–85. Publisher Full Text\n\nNetemeyer R, Bearden W, Sharma S: Scaling Procedures. Thousand Oaks, CA: SAGE Publications, Inc. 2003. Publisher Full Text\n\nBurnette JL: Implicit theories of body weight: entity beliefs can weigh you down. Pers Soc Psychol Bull. 2010; 36(3): 410–22. PubMed Abstract \| Publisher Full Text\n\nHan H, Glover GH, Jeong C: Cultural influences on the neural correlate of moral decision making processes. Behav Brain Res. 2014; 259: 215–28. PubMed Abstract \| Publisher Full Text\n\nHan H, Choi Y, Dawson KJ, et al.: Moral growth mindset is associated with change in voluntary service engagement. 2020. http://www.doi.org/10.17605/OSF.IO/AC6RH\n\nChiu C, Hong Y, Dweck CS: Toward an integrative model of personality and intelligence: A general framework and some preliminary steps. In: Sternberg RJ, Ruzgis P, editors. Personality and Intelligence. Cambridge, UK: Cambridge University Press. 1994; 104–34. Reference Source\n\nSchlenker BR, Chambers JR, Le BM: Conservatives are happier than liberals, but why? Political ideology, personality, and life satisfaction. J Res Pers. 2012; 46(2): 127–46. Publisher Full Text\n\nGraham J, Haidt J, Nosek BA: Liberals and conservatives rely on different sets of moral foundations. J Pers Soc Psychol. 2009; 96(5): 1029–46. PubMed Abstract \| Publisher Full Text\n\nGagne P, Hancock GR: Measurement Model Quality, Sample Size, and Solution Propriety in Confirmatory Factor Models. Multivariate Behav Res. 2006; 41(1): 65–83. PubMed Abstract \| Publisher Full Text\n\nFlora DB, Curran PJ: An empirical evaluation of alternative methods of estimation for confirmatory factor analysis with ordinal data. Psychol Methods. 2004; 9(4): 466–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHu L, Bentler PM: Cutoff criteria for fit indexes in covariance structure analysis: Conventional criteria versus new alternatives. Struct Equ Model A Multidiscip J. 1999; 6(1): 1–55. Publisher Full Text\n\nHong Y, Chiu C, Dweck CS, et al.: Implicit theories, attributions, and coping: A meaning system approach. J Pers Soc Psychol. 1999; 77(3): 588–99. Publisher Full Text\n\nChoi YJ, Han H, Dawson KJ, et al.: Measuring moral reasoning using moral dilemmas: evaluating reliability, validity, and differential item functioning of the behavioural defining issues test (bDIT). Eur J Dev Psychol. 2019; 16(5): 622–631. Publisher Full Text\n\nHan H, Dawson KJ, Thoma SJ, et al.: Developmental level of moral judgment influences behavioral patterns during moral decision-making. J Exp Educ. 2019. Publisher Full Text\n\nDavis MH: Measuring individual differences in empathy: Evidence for a multidimensional approach. J Pers Soc Psychol. 1983; 44(1): 113–26. Publisher Full Text\n\nAquino K, Reed A 2nd: The self-importance of moral identity. J Pers Soc Psychol. 2002; 83(6): 1423–40. PubMed Abstract \| Publisher Full Text\n\nMoore C, Detert JR, Klebe Trevino L, et al.: Why employees do bad things: Moral disengagement and unethical organizational behavior. Pers Psychol. 2012; 65(1): 1–48. Publisher Full Text\n\nBronk KC, Riches BR, Mangan SA: Claremont Purpose Scale: A Measure that Assesses the Three Dimensions of Purpose among Adolescents. Res Hum Dev. 2018; 15(2): 101–17. Publisher Full Text\n\nYeager DS, Trzesniewski KH, Tirri K, et al.: Adolescents' implicit theories predict desire for vengeance after peer conflicts: correlational and experimental evidence. Dev Psychol. 2011; 47(4): 1090–107. PubMed Abstract \| Publisher Full Text\n\nDiseth Å, Meland E, Breidablik HJ: Self-beliefs among students: Grade level and gender differences in self-esteem, self-efficacy and implicit theories of intelligence. Learn Individ Differ. 2014; 35: 1–8. Publisher Full Text\n\nTamir M, John OP, Srivastava S, et al.: Implicit theories of emotion: affective and social outcomes across a major life transition. J Pers Soc Psychol. 2007; 92(4): 731–44. PubMed Abstract \| Publisher Full Text\n\nHenseler J, Ringle CM, Sarstedt M: A new criterion for assessing discriminant validity in variance-based structural equation modeling. J Acad Mark Sci. 2015; 43(1): 115–35. Publisher Full Text\n\nYeager DS, Miu AS, Powers J, et al.: Implicit theories of personality and attributions of hostile intent: a meta-analysis, an experiment, and a longitudinal intervention. Child Dev. 2013; 84(5): 1651–67. PubMed Abstract \| Publisher Full Text\n\nLüftenegger M, Kollmayer M, Bergsmann E, et al.: Mathematically gifted students and high achievement: the role of motivation and classroom structure. High Abil Stud. 2015; 26(2): 227–43. Publisher Full Text\n\nPomerantz EM, Saxon JL: Conceptions of ability as stable and self-evaluative processes: a longitudinal examination. Child Dev. 2001; 72(1): 152–73. PubMed Abstract \| Publisher Full Text" }	[ { "id": "62240", "date": "27 Apr 2020", "name": "Michael T. Warren", "expertise": [ "Reviewer Expertise Developmental Psychology", "Moral 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\nHan and colleagues examined the psychometric properties of the English version of a growth mindset measure in the moral domain. Moral growth mindset (MGM) may prove to be a useful motivational construct in the scientific study of morality in general, and moral development in particular. I believe the authors present sufficient initial evidence of the validity and reliability of their measure, marking an important step in the scientific study of MGM in English-speaking populations.\n\nI see three major issues with the paper in its current form, and I would encourage the authors to revise their manuscript in light of my suggestions.\nFirst, my biggest concern is the use of phrases such as “improve one’s morals” and “improve your morals.” The former occurs in the paper’s conceptual framing (p. 3) and the latter appears in each of the MGM scale’s four items (p. 4). The issue with these phrases is that improving one’s morals seems to deviate conceptually from improving one’s morality. One might improve their morals by setting new (or higher) moral standards for themselves, yet they may fail miserably in living up to their moral values. By contrast, improving one’s morality involves actually becoming a better person, and this, I believe, is the construct the authors intended to measure. Since in my view the items miss the target to some extent, I have indicated that the work is only “partly” technically sound. Unfortunately, I don’t think much can be done about this issue at this point, but at a minimum I would recommend that the authors either provide an argument for the use of \"moral\" rather than \"morality\" in their scale, or identify this as a limitation of their scale. In addition, they might choose to argue that this concern is assuaged by the scale’s strong evidence of convergent validity with other measures of morality.\nSecond, I think the CFA results need to be communicated more fully, and that is why I have indicated that the statistical analyses and their interpretations are only “partly” appropriate. On p. 4, I certainly understand consulting previous studies (e.g., Han et al., 2018), but data from the current (i.e., English) study should be given primary importance in refining the English scale. I recommend reporting the factor loadings from the original CFA (i.e., before Items 1 and 2 were removed), so readers can evaluate whether removing these items was justified on empirical grounds. On a related note, I think it would be appropriate to acknowledge the small factor loading (-.39) for the reverse-scored item in Study 2.\n\nThird, I think the introduction to Study 2 (p. 5) should be expanded considerably. It would be very helpful to provide a brief rationale for why the selected constructs were chosen for convergent and discriminant validity testing. In addition, it would be helpful to specify hypotheses concerning the strength and direction of the associations between MGM and other constructs (i.e., with which constructs does MGM have strongest and weakest theoretical ties?), and why. The discussion currently states that the observed associations were “as hypothesized,” but no hypotheses were specified in the lead-up to Study 2. I also found myself wondering why Chiu et al.1 (1997)’s original 3-item English MGM measure was not included for convergent and incremental validity testing.\n\nMinor comments:\nPage 3: My understanding is that growth mindset generally concerns one’s beliefs about the malleability of one’s own (and others’) qualities. Thus, it seems a little bit too generic to define growth mindset as believing “it is possible to improve aspects of one’s life.” There are aspects of a person’s life (e.g., what kind of work they do; where they live, etc.) that are not qualities of their personhood. I suggest the authors consider revising their opening definition of growth mindset.\n\nPage 3: It’s not clear to me how allowing participants to define “moral” and “character” necessarily allows them to do so “without bias.” Instead, I think it would be more accurate to say that the approach taken leaves it up to participants to interpret \"moral\" and \"character\" according to their own subjective understandings of those terms. (Note that this approach makes no claim that participants’ understandings are “without bias.”)\n\nPages 3 and 5: I suggest the authors change the “Participants” heading to “Participants and procedures.”\n\nPage 4: I suggest confirming that three IRB approvals were needed for just two studies.\n\nPages 4-5: I suggest referring to model fit and reliability indices as either “indices” or “indexes,” rather than “indicators.” Given that CFA was involved, readers may assume “indicators” refers to measured variables loading onto latent factors.\n\nPage 5 (last paragraph of Study 1): I would like to suggest an alternative explanation as to why Items 1 and 2 (presumably) had lower factor loadings. These two were the only items to convey morality/character as dispositional (e.g., \"You have a certain morality and character...\"; \"Your morality and character are something about you...\"). By contrast, all items measured malleability beliefs, including the retained reverse-scored item (“To be honest, you can’t really improve your morals and character.”). My understanding is that a growth mindset is anchored in malleability beliefs, and having a growth mindset does not preclude the belief in moral dispositions (e.g., with effort I can become a more consistently/dispositionally honest person). In other words, perhaps the reason why Items 1 and 2 presumably had lower factor loadings was because they strayed somewhat from the core of the growth mindset construct (i.e., malleability beliefs), rather than because they used the vague qualifier, “much.” Just some food for thought.\n\nPage 5 (Participants section): Much of the first two paragraphs in this section is redundant with the procedures described in Study 1. The authors may wish to simply state that the same recruitment procedures were used as described in Study 1.\n\nPage 5: I would strongly urge the authors to omit the term, “marginally correlated” in relation to MGM’s association with the bDIT. Once a threshold for statistical significance has been set (e.g., .05), a finding is either statistically significant or non-significant. Correlations with p-values between .05 and .10 are non-significant.\n\nPage 6 (Table 3): I suggest indicating where Cronbach alphas are reported (i.e., on the diagonal).\n\nPage 6: More information on the potential utility of the MGM measure for understanding moral development would be a nice selling point for the scale. For example, this scale makes it possible to test whether MGM moderates the efficacy of moral education and social emotional learning interventions. The scale would also be an important outcome measure in examining how to nurture MGM (e.g., through process praise, teaching about neuroplasticity, etc.).\n\nPage 6: It is not yet clear why the authors would like to have conducted CFAs for the other measures. I would suggest they either drop this piece or further explain why additional CFAs would be desirable if the sample were large enough.\n\nPage 6: I think more explanation is needed as to why testing measurement invariance would be helpful. For example, the authors might say that examining measurement invariance across diverse groups of people (e.g., political conservatives vs. liberals; young adults vs. older adults) would help evaluate whether the scale—which leaves it up to participants to interpret morality and character in the item stems—in fact measures the same thing for groups who may use different underlying folk conceptions of morality.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "5484", "date": "07 May 2020", "name": "Hyemin Han", "role": "Author Response", "response": "Dear Dr. Warren, We sincerely appreciate your comments and suggestions on our manuscript. We found that they are very constructive and information. While revising our manuscript, we have done our best to address the concerns that you mentioned in your review report. Please find our responses to your comments below. Thank you very much for your time and consideration. Best, Hyemin Han (Corresponding author) Responses 1. First, my biggest concern is the use of phrases such as “improve one’s morals” and “improve your morals.” The former occurs in the paper’s conceptual framing (p. 3) and the latter appears in each of the MGM scale’s four items (p. 4). The issue with these phrases is that improving one’s morals seems to deviate conceptually from improving one’s morality. One might improve their morals by setting new (or higher) moral standards for themselves, yet they may fail miserably in living up to their moral values. By contrast, improving one’s morality involves actually becoming a better person, and this, I believe, is the construct the authors intended to measure. Since in my view the items miss the target to some extent, I have indicated that the work is only “partly” technically sound. Unfortunately, I don’t think much can be done about this issue at this point, but at a minimum I would recommend that the authors either provide an argument for the use of \"moral\" rather than \"morality\" in their scale, or identify this as a limitation of their scale. In addition, they might choose to argue that this concern is assuaged by the scale’s strong evidence of convergent validity with other measures of morality. Response: Thank you very much for your comment regarding the use of the terms in our study. In the revised manuscript, we added a paragraph describing why we decided to use “morals” instead of “morality” in our measure. The point is that we intended use the term to maintain the consistency with the prior study. “Finally, since Chiu et al. used terms related to specific morals and characteristics in their original three-item subscale (e.g., “A person’s moral character,” “whether a person is responsible and sincere,” “a person’s moral traits”), we decided to use “morals and character” in order to stay consistent with the construct they were measuring. That is, rather than measuring participants’ malleability beliefs about the overarching system of values they have, we wanted to measure malleability beliefs regarding individual morals, as did the original measure. Doing so may increase the chance for interventions since if people want to become a better person (improve their morality) they may need to believe that their values (morals) can be improved.” 2. Second, I think the CFA results need to be communicated more fully, and that is why I have indicated that the statistical analyses and their interpretations are only “partly” appropriate. On p. 4, I certainly understand consulting previous studies (e.g., Han et al., 2018), but data from the current (i.e., English) study should be given primary importance in refining the English scale. I recommend reporting the factor loadings from the original CFA (i.e., before Items 1 and 2 were removed), so readers can evaluate whether removing these items was justified on empirical grounds. On a related note, I think it would be appropriate to acknowledge the small factor loading (-.39) for the reverse-scored item in Study 2. Response: We appreciate your comment regarding how to report results from CFA. Following your suggestion, we added a supplementary table that demonstrates the factor loadings in 6-item and 5-item models. As you can see, Item 1 and Item 2 showed the lowest standardized factor loadings in the 6-item and 5-item models, respectively. In the revised manuscript, we mentioned the point that they were excluded from the measure due to their lowest standardized factor loadings. Table S1 Factor loadings from the CFA of the six- and five-item models in Study 1 Six-item model Five-item model Item Unstandardized Standardized Unstandardized Standardized You have certain morals and character, and you can’t really do much to improve it. -.71 -.58 - - Your morals and character are something about you that you can’t improve very much. .70 -.67 -.61 -.58 No matter who you are, you can significantly improve your morals and character. .64 .62 .68 .65 To be honest, you can’t really improve your morals and character. -.86 -.86 -.81 -.81 You can always substantially improve your morals and character. .66 .69 .72 .75 You can improve your basic morals and character considerably. .80 .'.83 .85 .88 Note: Bolded items (Items 1 and 2) were excluded from the finalized MGM measure. “In the supplementary table in Underlying data, we presented factor loadings for the six-item and five-item models. In the six-item model, Item 1 showed the lowest standardized factor loading, identical to what was reported in Han et al. (2018) 4. After excluding Item 1, Item 2 showed the lowest standardized loading in the five-item model, so we removed this item accordingly.” Moreover, we acknowledge the slightly low factor loading in Study 2: “However, it should be acknowledged that Item 4 showed a slightly lower factor loading in Study 2 compared with Study 1, although the overall model fit indices were excellent. This point might need to be tested in future studies with more samples.” 3. Third, I think the introduction to Study 2 (p. 5) should be expanded considerably. It would be very helpful to provide a brief rationale for why the selected constructs were chosen for convergent and discriminant validity testing. In addition, it would be helpful to specify hypotheses concerning the strength and direction of the associations between MGM and other constructs (i.e., with which constructs does MGM have strongest and weakest theoretical ties?), and why. The discussion currently states that the observed associations were “as hypothesized,” but no hypotheses were specified in the lead-up to Study 2. I also found myself wondering why Chiu et al. (1997)’s original 3-item English MGM measure was not included for convergent and incremental validity testing. Response: Thanks a lot for your suggestion regarding the expansion of the Study 2 introduction. Following your suggestion, in the revised manuscript, we elaborated the rationale regarding how the additional construct used in our study were selected with citations. Furthermore, in the methods section, per additional measurement, we explained the direction and effect size of the hypothesized correlation. “We selected several moral and positive psychological measures to test the convergent and divergent validity of the MGM measure. We employed the Implicit Theory Measure 1, which measures domain-general growth mindset and constitutes the basis of the MGM measure, to test convergent and discriminant validity. For the selection of moral psychological measures, we referred to recent articles about psychological constructs that significantly predict prosocial and civic behavior 31. They proposed moral judgment 18, 19, moral emotion (empathy) 20, and moral identity 21 as fundamental constructs in moral functioning. We also employed the Propensity to Morally Disengage Scale to examine whether the MGM showed negative correlation with moral disengagement 22 since Han et al. (2018) 4 reported that MGM promotes moral engagement. In addition to the aforementioned moral psychological measures, we used the Claremont Purpose Scale as a way to examine one’s positive development in terms of flourishing 23, given that purpose has been regarded as a possible moral virtue for eudemonic wellbeing 32. In general, according to the previous studies that examined the relationship between growth mindset, positive psychological indicators, and antisocial tendency (e.g., 24– 26), we hypothesized that the sizes of correlation coefficients between MGM and other indicators, except the general growth mindset, would be between .10 (small) and .30 (medium). We discussed further details regarding the hypothesized effect size of each measure in the following sections.” We agree with you that employing Chiu et al.’s original measure in the present study would be beneficial. However, we did not consider doing so because our measure was originally Based on Dweck’s updated six-item measure for general growth mindset. In the limitation section, we acknowledged your point for reader’s information. “Fourth, we did not employ Chiu et al.’s (1997) 5 original measure, which could be informative while conducting the convergent validity check, although our measure was based on Dweck’s (2000) 1 updated six-item general growth mindset measure.” 4. Page 3: My understanding is that growth mindset generally concerns one’s beliefs about the malleability of one’s own (and others’) qualities. Thus, it seems a little bit too generic to define growth mindset as believing “it is possible to improve aspects of one’s life.” There are aspects of a person’s life (e.g., what kind of work they do; where they live, etc.) that are not qualities of their personhood. I suggest the authors consider revising their opening definition of growth mindset. Response: We appreciate your suggestion regarding the introduction. We revised the introduction for a better definition of the growth mindset: “Growth mindset refers to the belief that it is possible to improve one’s abilities and qualities, such as intelligence or personality 1 . These individuals believe that this can be done through effort and learning, which helps fosters motivation. Higher motivation for those with a growth mindset is encouraged through having attitudes such as viewing hardships as a chance to work harder rather than an indication of failure, and striving for success due to genuinely wanting to learn instead of being concerned with how others view them 2” 5. Page 3: It’s not clear to me how allowing participants to define “moral” and “character” necessarily allows them to do so “without bias.” Instead, I think it would be more accurate to say that the approach taken leaves it up to participants to interpret \"moral\" and \"character\" according to their own subjective understandings of those terms. (Note that this approach makes no claim that participants’ understandings are “without bias.”) Response: We appreciate your comment regarding the use of the terms in our study. In the revised manuscript, we updated our explanation regarding the terms as per your comment: “Thus, we used “morals and characters” in order for participants to be able to define the terms based on their own experiences and understanding.” 6. Pages 3 and 5: I suggest the authors change the “Participants” heading to “Participants and procedures.” Response: Thanks a lot for your suggestion regarding the subsection. In the revised manuscript, following your and Dr. Mangan’s suggestions, we moved contents regarding the study procedures to a new subsection, “procedures.” 7. Page 4: I suggest confirming that three IRB approvals were needed for just two studies. Response: Thank you for your comment regarding the IRB numbers. In the revised manuscript, we clearly stated which IRB protocols are relevant to which specific study. 8. Pages 4-5: I suggest referring to model fit and reliability indices as either “indices” or “indexes,” rather than “indicators.” Given that CFA was involved, readers may assume “indicators” refers to measured variables loading onto latent factors. Response: We appreciate your comment regarding the use of the term. In the revised manuscript, as per your comment, we used “indices” in lieu of “indicators” while addressing CFA. 9. Page 5 (last paragraph of Study 1): I would like to suggest an alternative explanation as to why Items 1 and 2 (presumably) had lower factor loadings. These two were the only items to convey morality/character as dispositional (e.g., \"You have a certain morality and character...\"; \"Your morality and character are something about you...\"). By contrast, all items measured malleability beliefs, including the retained reverse-scored item (“To be honest, you can’t really improve your morals and character.”). My understanding is that a growth mindset is anchored in malleability beliefs, and having a growth mindset does not preclude the belief in moral dispositions (e.g., with effort I can become a more consistently/dispositionally honest person). In other words, perhaps the reason why Items 1 and 2 presumably had lower factor loadings was because they strayed somewhat from the core of the growth mindset construct (i.e., malleability beliefs), rather than because they used the vague qualifier, “much.” Just some food for thought. Response: Thanks a lot for the alternative explanation of the lower factor loadings of items 1 and 2. We added such an alternative explanation in the revised manuscript for readers’ information: “In addition, as another possibility, items 1 and 2 are more likely about entity beliefs, not malleability beliefs that constitute the basis of growth mindset. These items contain some words perhaps related to entity beliefs (e.g., “certain morals and characters...,” “something about you…”), so they might not directly measure the core of the growth mindset construct and showed lower factor loadings compared to the other items.” 10. Page 5 (Participants section): Much of the first two paragraphs in this section is redundant with the procedures described in Study 1. The authors may wish to simply state that the same recruitment procedures were used as described in Study 1. Response: Thank you very much for your suggestion for the brevity of our manuscript. We shortened the redundant part in Study 2 as per your suggestion. 11. Page 5: I would strongly urge the authors to omit the term, “marginally correlated” in relation to MGM’s association with the bDIT. Once a threshold for statistical significance has been set (e.g., .05), a finding is either statistically significant or non-significant. Correlations with p-values between .05 and .10 are non-significant. Response: Thanks a lot for your comment about the use of the term, “marginally correlated.” We agree with you that the use of the term is somehow inappropriate, so in the revised manuscript, we changed the part about interpreting the finding from correlation analysis: “The effect size of the correlation coefficient between MGM and bDIT was small as predicted, but the correlation was non-significant (p = .08).” 12. Page 6 (Table 3): I suggest indicating where Cronbach alphas are reported (i.e., on the diagonal). Response: We appreciate your suggestion. We added a brief description about where alphas were reported: “Cronbach αs are also reported (on the diagonal).” 13. Page 6: More information on the potential utility of the MGM measure for understanding moral development would be a nice selling point for the scale. For example, this scale makes it possible to test whether MGM moderates the efficacy of moral education and social emotional learning interventions. The scale would also be an important outcome measure in examining how to nurture MGM (e.g., through process praise, teaching about neuroplasticity, etc.). Response: Thanks a lot for your suggestion about the elaboration of the potential utility of the measure. In the introduction, we briefly mentioned how the measure could be used in moral education: “Additionally, if moral growth mindset motivates people to learn how to become more moral, as previous research suggests, then it is important for moral educators to have a tool to assess the malleability beliefs students have related to their morals. For example, if moral educators are able to identify that some students have a fixed mindset related to their morals, then an appropriate starting point may be to provide them with evidence that it is possible to improve moral character throughout one’s life.” 14. Page 6: It is not yet clear why the authors would like to have conducted CFAs for the other measures. I would suggest they either drop this piece or further explain why additional CFAs would be desirable if the sample were large enough. Response: We appreciate your comment regarding the CFA of additional measures. We dropped the part as per your comment since it was not essential in our study. 15. Page 6: I think more explanation is needed as to why testing measurement invariance would be helpful. For example, the authors might say that examining measurement invariance across diverse groups of people (e.g., political conservatives vs. liberals; young adults vs. older adults) would help evaluate whether the scale—which leaves it up to participants to interpret morality and character in the item stems—in fact measures the same thing for groups who may use different underlying folk conceptions of morality. Response: Thank you very much for your suggestion. We clarified the point in the revised manuscript: “To address this issue, measurement invariance test would be a way to examine whether the MGM measure, which allows participants to interpret “morals” and “character” by themselves, measures the same construct across different groups who may use different underlying folk conceptions of morals and character.”" } ] }, { "id": "62237", "date": "29 Apr 2020", "name": "Susan Mangan", "expertise": [ "Reviewer Expertise Developmental psychology", "positive psychology", "emerging adulthood", "positive pyschology interventions" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHan et al. suggest the creation of a moral growth mindset scale, which could make a significant contribution to the growing literature on growth mindset. The initial validity and reliability of this scale appears sound, and the inclusion of moral growth mindset (MGM) to the literature base seems evident. Despite the promise of this scale and the concept it captures, the article, as written, has some room for improvement. Most notably, the items themselves may benefit from further revision, more information is needed, especially on the various types of validity discussed in the article (e.g. convergent, divergent), and the writing could be clearer and more concise throughout.\nDetailed responses:\nLit review\nOne piece missing here is an argument for the significance of this scale. Why is moral growth mindset an important concept to measure? How does this measure add to existing literature (and existing measures on growth mindset + morality?) Were items for this scale taken from related scales (growth mindset?)\n\nStudy 1\nElaborate on the “Implicit theory measure.” Be sure to say 1-2 sentences about this to help us understand its significance to the current study. Participants\nHow were participants compensated? Class credit? Gift card? No compensation? (Ok, later in the next paragraph you mention class credit – I would mention in the first paragraph that participants were offered class credit to participate).\n\nTo help clarify each portion of the study and make it easy for readers to find the information that wish, I would add a heading of “Procedure” that begins with the “Participants received a link to the qulatircs survey…” Analysis\nWhat is “underlying data” and where is it located? From the final sentence of your analysis section.\n\nStudy 2\n“In study 2, we tested THE correlation between…” (Add “the” to sentence). You mention the SONA system and how participants selected studies here – make sure to also include this recruitment information in study 1. Additionally, here you include information about the order of survey scales and demographics, which is also not in study 1. In general, there is different information here than in study 1 – I would align these participant sections to include the same relevant information. I would also, again, separate this into a clear “participants” section and a clear “procedure” section.\nAdditionally: How long did it take participants to complete surveys? Were any attention check items included?\n\nMeasures\nWhile these measures look great – we need to understand their inclusion. In the literature review portion of this article there should be discussion of each construct and why/how you expect it to relate to moral growth mindset. Why are these good choices for convergent validity? You mention that further details are available in “extended data” but I don’t see a way to access this. Is all the relevant information explaining the connection of these scales to moral growth mindset provided there? Additionally, did you test for divergent validity? Content validity? Construct validity? If not, why? Ok, some indication of a test for discriminant validity is presented in analysis, but this should be listed with the other measures above. Similarly, more information is needed here about why you expect this to be divergent from moral growth mindset.\n\nDiscussion\n1st sentence: “We developed and tested….from youth participants.” These participants were emerging adults, correct? Not youth? I would elaborate heavily on this first paragraph. What does it mean that moral disengagement was negatively correlated with MGM? What does it indicate if MGM and growth mindset were discriminant? As this is a measure development paper, I would include a richer discussion of what you found and what it indicates for each type of validity. I’m not sure what you mean here in sentence two “In fact, the previous studies that developed and tested measurements for the mindset with diverse domains…” What is “the mindset”? Do you mean, that tested other types of domain-specific growth mindset? There is a lot of information about previous studies coming up here that should also be in the introduction/literature review section. This would be great to include so we know what you are expecting before the study is run. Then, here in the discussion, you can tell us if your predictions came out as expected or not. Paragraph 1 and 2 here are a little confusing. Be sure to start each paragraph with a general sentence that indicates what you found. Then, discuss each of your results that provides evidence for your statement, and conclude with a sentence that indicates to us the importance of these results. It feels like there are too many concepts included in each paragraph, and the writing is a bit confusing throughout. Last paragraph “However, there are limitationS” (missing s).\n\nItems\nThese items all feel a bit too similar – Each asks if morality can or cannot be “improved.” Did you consider other items with different word choices? For example, even “You can always become a more moral person with better character.” Or “It is possible to grow in your character and morality.” These items are so similar that it feels hard to argue any differences between them. Additionally, if possible it’s always best to include simpler words over more complicated ones. For instance “substantially” could easily be “a lot” and “considerably” could also be “a lot.” More complex words tax the participants a bit more, and may make the sentences more difficult to digest, especially for those with lower readings levels or from less educated backgrounds.\n\nGeneral notes\nThere are a few places where the writing could be condensed or the use of alternate terms would improve ease of reading. For instance, an example of condensing would be: in participants, “Participants were recruited from an undergraduate subject pool. The pool consisted of students who were enrolled in educational psychology classes” could be condensed to “Participants were recruited from students enrolled in educational psychology classes.” A for instance of somewhere where alternate terms would be benefitial would be in: Results, “First, all consistency indicators indicated…” Instead of using “indicator/indicated” here I would recommend “First, all consistency indicators revealed…” Or, in the spirit of condensing/being more specific, I might suggest: First, the measure demonstrated at least acceptable reliability according to both cronbach’s alpha values and test-retest reliability.”\nLook for these types of instances throughout the paper with an eye towards condensing repetitive language and becoming more specific.\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": "5485", "date": "07 May 2020", "name": "Hyemin Han", "role": "Author Response", "response": "Dear Dr. Mangan, We sincerely appreciate your comments and suggestions on our manuscript. We found that they are very constructive and information. While revising our manuscript, we have done our best to address the concerns that you mentioned in your review report. Please find our responses to your comments below. Thank you very much for your time and consideration. Best, Hyemin Han (Corresponding author) Responses 1. One piece missing here is an argument for the significance of this scale. Why is moral growth mindset an important concept to measure? How does this measure add to existing literature (and existing measures on growth mindset + morality?) Were items for this scale taken from related scales (growth mindset?) Response: We appreciate your comment regarding the explanation of why the measure is important. Also, we described how the items were developed. In the revised manuscript, we elaborated further details. “The results suggested that among younger populations, MGM might increase participants’ prosocial behavior due to the belief that it will make them morally better. Given this, MGM would be considered as a factor that contributes to moral development. In order to adequately examine how MGM contributes to moral development, however, it is necessary to have an appropriate measure. Additionally, if moral growth mindset motivates people to learn how to become more moral, as previous research suggests, then it is important for moral educators to have a tool to assess the malleability beliefs students have related to their morals. For example, if moral educators are able to identify that some students have a fixed mindset related to their morals, then an appropriate starting point may be to provide them with evidence that it is possible to improve moral character throughout one’s life.” “Instead, the inventors (HH, KJD, and YJC) of the Korean MGM measure created its English version based on the structure of the Korean version and the wording in the Implicit Theory measure. In addition, the Implicit Theory measure was used due to the fact that it had six items and was based on Dweck’s original measure of growth mindset for intelligence. As a result, the tested measure included six items as well (e.g., “No matter who you are, you can significantly improve your morals and character”) and answers were anchored to a six-point Likert scale (see Extended data for the full measure 10 ).” 2. Elaborate on the “Implicit theory measure.” Be sure to say 1-2 sentences about this to help us understand its significance to the current study. Response: Thank you very much for your suggestion regarding the elaboration of the construct. We elaborated such a point in the revised manuscript: “Growth mindset refers to the belief that it is possible to improve one’s abilities and qualities, such as intelligence or personality 1 . These individuals believe that this can be done through effort and learning, which helps fosters motivation. Higher motivation for those with a growth mindset is encouraged through having attitudes such as viewing hardships as a chance to work harder rather than an indication of failure, and striving for success due to genuinely wanting to learn instead of being concerned with how others view them 2” 3. How were participants compensated? Class credit? Gift card? No compensation?(Ok, later in the next paragraph you mention class credit – I would mention in the first paragraph that participants were offered class credit to participate). Response: Thanks a lot for your request for the clarification of the compensation. In the revised manuscript, such a point is more clearly stated: “Participants were recruited from students enrolled in undergraduate educational psychology classes and they were provided with a course credit.” 4. To help clarify each portion of the study and make it easy for readers to find the information that wish, I would add a heading of “Procedure” that begins with the “Participants received a link to the qulatircs survey…” Response: We sincerely appreciate your suggestion regarding the use of the “procedure” subsection. In the revised manuscript, we created the new subsection for a better structure. 5. What is “underlying data” and where is it located? From the final sentence of your analysis section. Response: Thank you for your comment regarding “underlying data.” “Underlying data” is a way to include supplementary materials in F1000Research. Readers can download the supplementary materials with the URL provided at the end of the main text. More specifically, a link to an open science repository is provided in the “data statement” section as per the journal guidelines. 6. “In study 2, we tested THE correlation between…” (Add “the” to sentence).You mention the SONA system and how participants selected studies here – make sure to also include this recruitment information in study 1. Additionally, here you include information about the order of survey scales and demographics, which is also not in study 1. In general, there is different information here than in study 1 – I would align these participant sections to include the same relevant information. I would also, again, separate this into a clear “participants” section and a clear “procedure” section. Response: We appreciate your comments regarding the typo and the use of the independent subsection, “procedure.” We addressed these issues in the revised manuscript. 7. Additionally: How long did it take participants to complete surveys? Were any attention check items included? Response: Thanks a lot for your kind comment regarding the survey duration. Unfortunately, we could not include any attention check items in the survey form. We explained further details in the limitation section: “Third, although participants spent about 33.98 minutes (median) to complete Study 2 we did not include any attention check items.” 8. While these measures look great – we need to understand their inclusion. In the literature review portion of this article there should be discussion of each construct and why/how you expect it to relate to moral growth mindset. Why are these good choices for convergent validity? Response: We sincerely appreciate your comment regarding the rationale for the inclusion of the additional measures. In the introduction section in Study 2, we added explanations regarding the point. In addition, while describing each additional measure, we explained the rational for the inclusion as well as the hypothesized correlation with MGM. “We selected several moral and positive psychological measures to test the convergent and divergent validity of the MGM measure. We employed the Implicit Theory Measure 1, which measures domain-general growth mindset and constitutes the basis of the MGM measure, to test convergent and discriminant validity. For the selection of moral psychological measures, we referred to recent articles about psychological constructs that significantly predict prosocial and civic behavior 31. They proposed moral judgment 18, 19, moral emotion (empathy) 20, and moral identity 21 as fundamental constructs in moral functioning. We also employed the Propensity to Morally Disengage Scale to examine whether the MGM showed negative correlation with moral disengagement 22 since Han et al. (2018) 4 reported that MGM promotes moral engagement. In addition to the aforementioned moral psychological measures, we used the Claremont Purpose Scale as a way to examine one’s positive development in terms of flourishing 23, given that purpose has been regarded as a possible moral virtue for eudemonic wellbeing 32. In general, according to the previous studies that examined the relationship between growth mindset, positive psychological indicators, and antisocial tendency (e.g., 24– 26), we hypothesized that the sizes of correlation coefficients between MGM and other indicators, except the general growth mindset, would be between .10 (small) and .30 (medium). We discussed further details regarding the hypothesized effect size of each measure in the following sections.” 9. You mention that further details are available in “extended data” but I don’t see a way to access this. Is all the relevant information explaining the connection of these scales to moral growth mindset provided there? Response: Thank you for your comment regarding the “extended data.” Same to the “underlying data,” “extended data” can also be downloaded with the link provided at the end of the main text. In the revised manuscript, as we mentioned in our response to your comment above, we explained further details regarding each additional measure in the methods section in Study 2. 10. Additionally, did you test for divergent validity? Content validity? Construct validity? If not, why? Ok, some indication of a test for discriminant validity is presented in analysis, but this should be listed with the other measures above. Similarly, more information is needed here about why you expect this to be divergent from moral growth mindset. Response: We appreciate your comment regarding the clarification of the tests that we conducted. In the revised manuscript, we explained further details. “We aimed at testing the validity of the measure, construct, convergent, and divergent validity. We selected several moral and positive psychological measures to test the convergent and divergent validity of the MGM measure. We employed the Implicit Theory Measure 1, which measures domain-general growth mindset and constitutes the basis of the MGM measure, to test convergent and discriminant validity. For the selection of moral psychological measures, we referred to recent articles about psychological constructs that significantly predict prosocial and civic behavior 31. They proposed moral judgment 18, 19, moral emotion (empathy) 20, and moral identity 21 as fundamental constructs in moral functioning. We also employed the Propensity to Morally Disengage Scale to examine whether the MGM showed negative correlation with moral disengagement 22 since Han et al. (2018) 4 reported that MGM promotes moral engagement. In addition to the aforementioned moral psychological measures, we used the Claremont Purpose Scale as a way to examine one’s positive development in terms of flourishing 23, given that purpose has been regarded as a possible moral virtue for eudemonic wellbeing 32.” “Given that the Implicit Theory Measure measures one’s general growth mindset, we expected that it would be positively correlated with MGM. However, because the construct measured by the Implicit Theory Measure is not domain specific, we also expected that the MGM would not completely overlap with this construct (discriminant validity). Given these, the effect size of the correlation coefficient would be medium to large (r = +.3 - +.5).” 11. 1st sentence: “We developed and tested….from youth participants.” These participants were emerging adults, correct? Not youth? Response: Thanks for your point regarding the correct use of the term. Yes, that is correct. So, we used “emerging adults” instead of “youth…” in the revised manuscript: “We developed and tested the English version of the MGM measure in this study with data collected from emerging adult participants.” 12. I would elaborate heavily on this first paragraph. What does it mean that moral disengagement was negatively correlated with MGM? What does it indicate if MGM and growth mindset were discriminant? As this is a measure development paper, I would include a richer discussion of what you found and what it indicates for each type of validity. Response: We appreciate your comment regarding moral disengagement. In the section that describes moral disengagement and its measure, we address the points that you mentioned: “Propensity to Morally Disengage Scale. The moral disengagement scale measures one’s propensity to disengage from moral behavior within morally problematic situations 22. It measures moral disengagement propensities for eight mechanisms (i.e., moral justification, euphemistic labeling, advantageous comparison, displacement of responsibility, diffusion of responsibility, distortion of consequences, dehumanization, attribution of blame) with eight items (one item per mechanism). We used a composite score of the eight items. The internal structure of the scale was tested with CFA by Moore et al. (2012) 22. As Bandura (2002) proposed 35, moral disengagement is negatively associated with motivation for moral engagement. Thus, we expected moral disengagement would be negatively associated with MGM while the effect size of the correlation would be similar to the cases of the IRI and MIS (small to medium; r = -.1 - .3).” In addition, in the discussion section, we elaborated the meaning of the negative correlation found from our analysis: “In addition, moral disengagement was negatively correlated with MGM. Since moral disengagement allows people to dismiss negative feelings, they may have about behaving immorally using the eight mechanisms previously mentioned, this increases the likelihood of continuing to behave immorally. In this way, moral disengagement and MGM have somewhat reverse trajectories. As hypothesized, this suggests that MGM may promote engaging in moral behavior. In addition, since moral internalization, which has been shown to inhibit moral disengagement 39, was also positively correlated with MGM, it makes sense that our measure was negatively correlated with moral disengagement. If somebody has a strong sense of their morals and these values are internalized, this may help them to stay engaged with their standards and furthermore, be motivated to continue to be morally better.” 13. I’m not sure what you mean here in sentence two “In fact, the previous studies that developed and tested measurements for the mindset with diverse domains…” What is “the mindset”? Do you mean, that tested other types of domain-specific growth mindset? Response: Thank you for your request for the clarification. Yes, that is correct. In the revised manuscript, we specified the nature of the mindset: “In fact, the previous studies that developed and tested measurements for diverse types of domain-specific growth mindset have shown that the measurements possessed good reliability and validity as well (e.g., 29, 30).” 14. There is a lot of information about previous studies coming up here that should also be in the introduction/literature review section. This would be great to include so we know what you are expecting before the study is run. Then, here in the discussion, you can tell us if your predictions came out as expected or not. Response: We sincerely appreciate your suggestion regarding rewriting the introduction. We agree with you that some theoretical contents that were presented in the discussion section in the original manuscript could be moved on to the introduction section for a better structure. Following your suggestion, in the revised manuscript, we presented such contents in the general introduction or introduction of each study. 15. Paragraph 1 and 2 here are a little confusing. Be sure to start each paragraph with a general sentence that indicates what you found. Then, discuss each of your results that provides evidence for your statement, and conclude with a sentence that indicates to us the importance of these results. It feels like there are too many concepts included in each paragraph, and the writing is a bit confusing throughout. Response: We appreciate your comment regarding the discussion section. As you suggested, in the revised manuscript, we slightly restructured the paragraphs in the discussion section. We revised each of following paragraphs so that it discusses one specific point each time. 16. Last paragraph “However, there are limitationS” (missing s). Response: Thank you very much for your comment on the typo. We corrected the point in the revised manuscript. 17. These items all feel a bit too similar – Each asks if morality can or cannot be “improved.” Did you consider other items with different word choices? For example, even “You can always become a more moral person with better character.” Or “It is possible to grow in your character and morality.” These items are so similar that it feels hard to argue any differences between them. Additionally, if possible it’s always best to include simpler words over more complicated ones. For instance “substantially” could easily be “a lot” and “considerably” could also be “a lot.” More complex words tax the participants a bit more, and may make the sentences more difficult to digest, especially for those with lower readings levels or from less educated backgrounds. Response: We appreciate your comments regarding the items used in our measure. Yes, we agree with you that some words used in the items are somehow complex to be easily understood by younger participants. So, we also think that such items may need to be modified if the measure is to be administrated among younger populations. Also, we also acknowledged that some words (e.g., “improve”) were repeatedly used in multiple items. Since we intended to keep the consistency with the original measures that we referred to (e.g., Dweck’s general growth mindset measure, the Korean version of the MGM measure), we ended up with using such terms in our measure. We explained these points in the limitation section: “Fifth, the items used in the MGM measure could be revised particularly when being administered among younger populations. We decided to use the current wordings to maintain consistency with the Korean version of the MGM measure and the Implicit Theory Measure, which constituted the basis of our measure. However, to make the measure more applicable to younger populations, some complex words (e.g., “substantially,” “considerably”) could be replaced with simpler words (e.g., “a lot”). Finally, since several items in the measure might seem to be similar, the words could be revised in future studies, particularly those focusing on children or young adolescents.” 18. There are a few places where the writing could be condensed or the use of alternate terms would improve ease of reading. For instance, an example of condensing would be: in participants, “Participants were recruited from an undergraduate subject pool. The pool consisted of students who were enrolled in educational psychology classes” could be condensed to “Participants were recruited from students enrolled in educational psychology classes.” A for instance of somewhere where alternate terms would be benefitial would be in: Results, “First, all consistency indicators indicated…” Instead of using “indicator/indicated” here I would recommend “First, all consistency indicators revealed…” Or, in the spirit of condensing/being more specific, I might suggest: First, the measure demonstrated at least acceptable reliability according to both cronbach’s alpha values and test-retest reliability.” Look for these types of instances throughout the paper with an eye towards condensing repetitive language and becoming more specific. Response: We sincerely appreciate your suggestions regarding the brevity of the manuscript. We agree with you that increasing the brevity is essential to enable potential readers to better understand the overall theme of our manuscript while saving their time. Thus, we edited the whole manuscript during the current revision process. In addition, we revised the manuscript as an effort to minimize repeating to use the same words in the multiple places." } ] } ]	1	https://f1000research.com/articles/9-256
https://f1000research.com/articles/9-274/v1	20 Apr 20	{ "type": "Correspondence", "title": "Comment on Raine (2019) ‘The neuromoral theory of antisocial, violent, and psychopathic behavior’", "authors": [ "Hyemin Han" ], "abstract": "Raine (2019) reviewed previous research on the neural correlates of antisocial, violent, and psychopathic behavior based on previous studies of neuroscience of morality. The author identified neural circutries associated with the aforementioned types of antisocial behaviors. However, in the review, Raine acknowledged a limitation in his arguments, the lack of evidence supporting the presence of the neural circutries. In this correspondence, I intend to show that this limitation can be addressed with additional evience from recent neuroimaging research and the evidence can support the presence of the neural circutiries of antisociality proposed by Raine.", "keywords": [ "psychopathy", "antisociality", "morality", "moral psychology", "neuroscience" ], "content": "\n\nA review article by Raine was published in Psychiatry Research in 2019 concerning neuromoral theory of antisocial, violent, and psychopathic behavior1. The author proposed a comprehensive model of the neural network of morality and antisociality to explain the neural-level mechanisms of antisocial behavior. The author referred to previous neuroimaging studies and meta-analyses to identity the aforementioned neural networks and proposed that the prefrontal cortex, amygdala, insula, and anterior cingulate cortex are included in both networks, while the striatum is included in the antisociality network. The author stated two limitations regarding the network model that he proposed: first, the involvement of the insula and cingulate cortex regions in the neural networks could not be sufficiently supported by previous neuroimaging studies and meta-analyses; second, evidence that supports the involvement of the striatum in the morality network is insufficient.\n\nAlthough Raine raised the aforementioned two concerns regarding the lack of supporting evidence, I suggest that recent research in the field of social neuroscience can address them. Herein, I introduce two supporting findings, one from online-based large-scale analysis of neuroimaging data, and the other from recent neuroimaging experiments focusing on brain circuitries associated with morality. These recent research findings will be able to provide evidence of the involvement of the insula, cingulate cortex, and striatum regions in the neuromoral circuits.\n\nFirst, a result from large-scale meta-analysis of previous neuroimaging studies provides evidence supporting Raine’s model. Thanks to the development of information technology, performing meta-analysis of large-scale neuroimaging data has become feasible. A recently invented web-based meta-analysis tool, NeuroSynth, is one example2. NeuroSynth automatically gathers coordinate information that is reported in published neuroimaging articles and performs meta-analysis of the gathered information. A result from a meta-analysis of 87 studies and 2,806 activation foci that are associated with a keyword “moral” demonstrates that the left insula and anterior and posterior cingulate cortices show significant common activity across moral task conditions (see http://neurosynth.org/analyses/terms/moral/ for further details). This result provides evidence that supports the involvement of the insula and cingulate cortices in the neural network of morality based on large-scale data. In fact, the three meta-analysis articles that Raine reviewed meta-analyzed relatively fewer numbers of neuroimaging studies (references3–5), so he could only tentatively propose the involvement of the insula and cingulate cortex regions in the neuromoral network. Hence, I suggest that the Raine’s argument is supported by large-scale neuroimaging data and the result from NeuroSynth analysis. Moreover, the reported involvement of the left insula may suggest the possibility of laterality effects that Raine mentioned in his review, although more research that directly focuses on the laterality effects should be conducted.\n\nSecond, recent neuroimaging studies by my research group6–8 suggest that the insula and striatum regions showed significant activation and interaction with prefrontal and cingulate regions, which were indicated as core regions in the neural network of morality by Raine, in moral task conditions. The author tentatively proposed that increasing evidence may suggest that the striatum can be included in the morality network as well as the antisociality network. The new neuroimaging studies may provide additional evidence that supports the involvement of the striatum in the morality network. Our neuroimaging study from 20146 reported that the insula, cingulate cortex, and striatum (e.g. caudate and putamen) were significantly activated when participants were solving moral dilemmas (see Table S1 in 6 for further details). Such findings were also supported by a recent reanalysis with Bayesian inference7. Furthermore, our study from 20168 conducted psychophysiological interaction analysis and connectivity analysis based on Granger causality to examine interactions and connections among brain regions in moral task conditions. This study reported that the insula and striatum regions significantly interacted and were connected with other morality-related regions including the medial prefrontal and cingulate cortices. I suggest these findings can also support Raine’s argument regarding the role of the insula in the neural network of morality as well as his tentative proposal regarding the involvement of the striatum in the same network. In particular, the second study7 provides more direct evidence that supports the presence of the network because it used analysis methods that were designed to examine interaction and connectivity between different brain regions.\n\nGiven the aforementioned additional large-scale analysis and neuroimaging studies, the insula, cingulate cortex, and striatum regions can be considered as parts of the neural network of morality. I conclude that Raine’s argument about the neural network of antisociality that he proposed, with some reservations due to lack of evidence, can be well supported by the analyses that I introduce here.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "References\n\nRaine A: The neuromoral theory of antisocial, violent, and psychopathic behavior. Psychiatry Res. 2019; 277: 64–9. PubMed Abstract \| Publisher Full Text\n\nYarkoni T, Poldrack RA, Nichols TE, et al.: Large-scale automated synthesis of human functional neuroimaging data. Nat Methods. 2011; 8(8): 665–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHan H: Neural correlates of moral sensitivity and moral judgment associated with brain circuitries of selfhood: A meta-analysis. J Moral Educ. 2017; 46(2): 97–113. Publisher Full Text\n\nEres R, Louis WR, Molenberghs P: Common and distinct neural networks involved in fMRI studies investigating morality: an ALE meta-analysis. Soc Neurosci. 2018; 13(4): 384–398. PubMed Abstract \| Publisher Full Text\n\nGarrigan B, Adlam AL, Langdon PE: The neural correlates of moral decision-making: A systematic review and meta-analysis of moral evaluations and response decision judgements. Brain Cogn. 2016; 108: 88–97. PubMed Abstract \| Publisher Full Text\n\nHan H, Glover GH, Jeong C: Cultural influences on the neural correlate of moral decision making processes. Behav Brain Res. 2014; 259: 215–28. PubMed Abstract \| Publisher Full Text\n\nHan H, Park J: Using SPM 12’s Second-level Bayesian Inference Procedure for fMRI Analysis: Practical Guidelines for End Users. Front Neuroinform. 2018; 12: 1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHan H, Chen J, Jeong C, et al.: Influence of the cortical midline structures on moral emotion and motivation in moral decision-making. Behav Brain Res. 2016; 302: 237–51. PubMed Abstract \| Publisher Full Text" }	[ { "id": "62632", "date": "12 May 2020", "name": "Ji-Won Hur", "expertise": [ "Reviewer Expertise Psychopathology", "Social neuroscience." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe current literature is a well written concise commentary. The title and the abstract precisely summarize the content of the article. The authors provided evidence supporting Raine’s model on the presence of the neural circuitry of antisociality. The concluding paragraph also clearly reflects the author's argument. This commentary would be useful to design or establish further research or hypothetical model regarding the insula, cingulate cortex, and striatum regions as parts of the neural network of morality or antisociality. (there are some typos (circutries) in the manuscript).\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Yes\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? Yes\n\nIs the conclusion balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "5736", "date": "21 Jul 2020", "name": "Hyemin Han", "role": "Author Response", "response": "Thank you very much for your kind comments. First of all, I corrected the typos in the manuscript that you mentioned. Second, following your suggestion and other reviewers' points, I added some points related to how my commentary can provide insights about how to develop further research questions and hypotheses." } ] }, { "id": "64888", "date": "07 Jul 2020", "name": "Pascal Molenberghs", "expertise": [ "Reviewer Expertise fMRI", "morality", "meta-analyses" ], "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 author provides a comment on a recently published article by Raine (2019) which is about a neuromodal theory of antisocial, violent, and psychopathic behavior.\n\nMajor comments:\n\nThe author makes two main claims:\nThe first claim is about the insula and cingulate cortex involvement in morality. According to the author, there is evidence that the insula and cingulate cortex are involved in morality based on a meta-analysis in Neurosynth with the word “moral”. It is stated that: “A result from a meta-analysis of 87 studies and 2,806 activation foci that are associated with a keyword “moral” demonstrates that the left insula and anterior and posterior cingulate cortices show significant common activity across moral task conditions (see http://neurosynth.org/analyses/terms/moral/ for further details).”\nI had a look at the result provided by the link, but I couldn’t see much evidence of insula of cingulate cortex involvement. Maybe there is a voxel or two that overlaps with the left insula and cingulate cortex but there is barely any evidence of this. Also, although I like the program Neurosynth, its data is quite messy because it just automatically scans studies based on a word (in this case “moral”). The algorithm will often make mistakes that can be overcome by a carefully conducted “manual” (i.e., not automatic) neuroimaging meta-analysis. Therefore, I wouldn’t trust a Neurosynth meta-analysis more than a thoroughly conducted manual neuroimaging meta-analyses on the topic. Given that several manual neuroimaging meta-analyses on the topic of morality couldn’t find any evidence of insula and cingulate cortex involvement, I don’t think that this Neurosynth meta-analysis (that barely shows any evidence of insula or cingulate cortex activity) is now the crucial missing evidence that we have been waiting for.\n\nThe second claim is that the striatum is involved in the morality network. Here the author uses three neuroimaging studies by his own research group. The three studies seem to be based on the same data of around 16 (sic) participants. I don’t doubt that the author is familiar with their own study and that striatum was activated but again, I don’t think this is the convincing evidence to finally claim that the striatum is consistently involved in morality. If the author wants to make the point about the striatum (or the insula and cingulate cortex), they should do a thorough look through the neuroimaging morality literature to argue their case.\n\nIn short, I don’t think the comment adds much to the literature.\n\nMinor comments:\n1) circutiries => circuitries 2) recently invented => Neurosynth has been around since 2011\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Yes\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? No\n\nIs the conclusion balanced and justified on the basis of the presented arguments? No", "responses": [] }, { "id": "65587", "date": "15 Jul 2020", "name": "Fernando Barbosa", "expertise": [ "Reviewer Expertise Psychopathy", "Antisocial Behavior", "EEG/ERP", "fMRI", "Neuropsychology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author sought to provide additional support concerning the neuronal bases of the neuromoral model of antisocial behavior, initially proposed by Raine and Yang (2006) and recently updated by Raine (2019), who reviewed findings on brain mechanisms underlying moral decision-making and antisocial behavior that were published after the initial proposal. Specifically, this comment was intended to provide evidence of the involvement of the insula, cingulate cortex, and striatum in the circuits of the neuromoral model. To that end, the author uses: (a) NeuroSynth (which is not recent) to obtain meta-analytic data from neuroimaging studies of (supposedly) moral decision-making; and (b) results from his own neuroimaging studies focusing on brain circuitries associated with morality.\nAlthough the NeuroSynth-based meta-analysis involved 87 studies, it should be noted that: (a) the NeuroSynth performs a semantic search limited to the abstracts of the papers; (b) many of the said studies do not report the involvement of the insula or the cingulate cortex in moral tasks; (c) the NeuroSynth lacks specificity, as moral decision-making is often studied with other constructs, which may have induced the observed brain activity; and (d) automated meta-analyses prevent the careful examination of relevant publication indices, such as the publication/confirmation bias. For these reasons and taking the NeuroSynth results into account, which do not seem substantial per se, it is highly questionable whether the author provides stronger evidence than the one compiled by Raine in his own review to support his cautious arguments. It is worth mentioning that Raine refers to four meta-analyses, and most support the role of the cingulate in the neuromoral circuit, but he also explains that a recent meta-analysis by Eres et al. (2018) does not.\n\nSimilarly, Raine reports meta-analytic studies that implicate the insula in morality, but he also explains that other meta-analyses do not (one of them was conducted by the author of this comment), leading to the conclusion that evidence is not sufficient to consider the insula as playing a role in moral decision-making. Moreover, given the fact that recent meta-analyses of morality have not directly implicated the striatum (again, one was authored by Han, 2017), Raine judiciously considers its inclusion in the neuromoral model debatable. The studies conducted and reported by the author of this comment do lend additional support to the possible role of the insula and striatum in moral decision-making, but such results do not provide stronger evidence than the meta-analytic findings compiled by Raine.\n\nSumming-up, the arguments and results presented here do not make the findings regarding the implication of the cingulate, insula and striatum in moral decision-making any more consistent, nor any less debatable. Both the claim that “the insula, cingulate cortex, and striatum can be considered as parts of the neural network of morality”, and the conclusion that such claim “can be well supported by the analyses that I [the author] introduce here” sound like overstatements. Raine’s cautiousness is still well justified.\n\nMinor issues: “circutries”, circutiries, and “evience” are typos.\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Yes\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? Partly\n\nIs the conclusion balanced and justified on the basis of the presented arguments? Partly", "responses": [ { "c_id": "5735", "date": "21 Jul 2020", "name": "Hyemin Han", "role": "Author Response", "response": "1. Although the NeuroSynth-based meta-analysis involved 87 studies, it should be noted that: (a) the NeuroSynth performs a semantic search limited to the abstracts of the papers; (b) many of the said studies do not report the involvement of the insula or the cingulate cortex in moral tasks; (c) the NeuroSynth lacks specificity, as moral decision-making is often studied with other constructs, which may have induced the observed brain activity; and (d) automated meta-analyses prevent the careful examination of relevant publication indices, such as the publication/confirmation bias. For these reasons and taking the NeuroSynth results into account, which do not seem substantial per se, it is highly questionable whether the author provides stronger evidence than the one compiled by Raine in his own review to support his cautious arguments. It is worth mentioning that Raine refers to four meta-analyses, and most support the role of the cingulate in the neuromoral circuit, but he also explains that a recent meta-analysis by Eres et al. (2018) does not. Response: Thank you very much for your comments regarding the limitations of NeuroSynth and the point related to the involvement of the cingulate cortex presented in the meta-analysis. In the revised manuscript, I added some descriptions about the limitations of NeuroSynth that warrant a cautious interpretation of results. Simultaneously, as a way to partially address the issue, I introduced NeuroQuery that analyzes the full article texts with Machine learning: Moreover, a recently developed analysis tool, NeuroQuery 9, also reported the consistent result. Unlike NeuroSynth, which only analyzes published papers’ abstracts, NeuroQuery analyzes the full article texts, so it shows better selectivity compared with NeuroSynth 9. It employs machine learning to examine the pattern of neural activation associated with a keyword of interest 9. When “morality” was explored by NeuroQuery, both the left and right insula and cingulate cortex showed strong association with the keyword across 68 studies (see https://neuroquery.org/query?text=morality for further details). Furthermore, I added a paragraph that discusses some additional caveats while interpreting the results from the large-scale analyses and meta-analyses: However, there are several caveats while interpreting these findings. First, although the involvement of the insula was consistently supported by two large-scale neuroimaging data analyses, some of the introduced meta-analyses did not report such a result. One point related to the large-scale analyses that should be considered is that compared with meta-analyses that were conducted with human-involved literature review, such automated analyses, particularly NeuroSynth, simple analyze texts per se so their selectivity might not be optimal. This point warrants further studies that more carefully explore large-scale database. Second, none of the meta-analyses and NeuroSynth directly demonstrated the association between the striatum with morality tasks. This might be due to that the analyzed previous studies were mainly about morality, not antisociality, so they might not be able to well address the neural correlates of antisociality.2. Similarly, Raine reports meta-analytic studies that implicate the insula in morality, but he also explains that other meta-analyses do not (one of them was conducted by the author of this comment), leading to the conclusion that evidence is not sufficient to consider the insula as playing a role in moral decision-making. Moreover, given the fact that recent meta-analyses of morality have not directly implicated the striatum (again, one was authored by Han, 2017), Raine judiciously considers its inclusion in the neuromoral model debatable.The studies conducted and reported by the author of this comment do lend additional support to the possible role of the insula and striatum in moral decision-making, but such results do not provide stronger evidence than the meta-analytic findings compiled by Raine.Response: I sincerely appreciate your comments regarding the involvement of the insula and striatum in the circuitries. I added some points from NeuroQuery while toning down my interpretations in general. In addition to these neuroimaging experiments, the result from NeuroQuery might also provide additional supports. Although the size of the identified region was small, NeuroQuery reported that when “morality” was entered, a part of the right putamen, which constitutes the dorsal striatum, showed a significant association with the keyword. Several previous fMRI studies have shown that the putamen was associated with intuition-related moral judgment 6, evaluation of moral intentionality of an actor in the relation with potential benefits and losses 10, and modulation of moral behavior 11. Hence, future studies may focus on the putamen as a possible candidate region to be included in the morality-antisociality circuitries based on these previous findings. Furthermore, although additional investigations with more focused experimental designs are warranted, such findings might be able to provide ideas about the involvement of striatal regions, particularly the putamen, in the morality-antisociality network.3. Summing-up, the arguments and results presented here do not make the findings regarding the implication of the cingulate, insula and striatum in moral decision-making any more consistent, nor any less debatable. Both the claim that “the insula, cingulate cortex, and striatum can be considered as parts of the neural network of morality”, and the conclusion that such claim “can be well supported by the analyses that I [the author] introduce here” sound like overstatements. Raine’s cautiousness is still well justified.Response: Thank you very much for your comments regarding the overall tone of my commentary. I agree with you that the limitations in the existing literature cannot completely confirm the involvement of the aforementioned regions in the circuitries. Thus, I toned down the introduction and conclusion accordingly. Instead of strongly argue that every single issue can be addressed by additional evidence, I proposed that the evidence is somehow suggestive. In other words, I proposed several directions for future research based on the added evidence. Also, as a way to strength my point, I additionally introduced NeuroQuery as well. These recent research findings will be able to provide evidence of the involvement of the insula and cingulate cortex, which are involved in both moral and antisocial brain circuitries. Furthermore, some additional evidence might be able to support the involvement of parts of striatal regions in the circuit of antisociality although the evidence is not sufficient to completely address the issue. At least, the additional evidence might be able to provide ideas about potential research questions and hypotheses focusing on the striatum in morality and antisociality. Given the aforementioned additional large-scale analyses and neuroimaging studies, we might have additional evidence that can support the point that the insula and cingulate cortex can be considered as parts of the neural network of morality. Particularly, both large-scale analyses, NeuroSynth and NeuroQuery, supported such a point although the meta-analyses introduced in Raine’s article demonstrated mixed results. One remaining issue is that although the association between the moral-antisocial circuitry and the striatum was partially supported by the additional neuroimaging experiments and NeuroQuery, NeuroSynth and the majority of the meta-analyses did not report such a result. Even if that is the case, the aforementioned results related to the functioning of the striatum might be able to provide researchers with some ideas for research question and hypothesis building. Given that the involvement of the striatum, particularly the putamen, was partially supported, future neuroimaging studies that intend to explore the antisocial circuitry may focus the point with more specialized research designs.4. Minor issues: “circutries”, circutiries, and “evience” are typos. Response: I appreciate your comments about the typos. In the revised manuscript, I corrected those points." } ] } ]	1	https://f1000research.com/articles/9-274
https://f1000research.com/articles/9-752/v1	21 Jul 20	{ "type": "Research Article", "title": "Effects of aflatoxin B1 on the submandibular salivary gland of albino rats and possible therapeutic potential of Rosmarinus officinalis: a light and electron microscopic study", "authors": [ "Bassant Ashraf", "Dahlia Ghazy", "Mohamed Shamel", "Bassant Ashraf", "Dahlia Ghazy" ], "abstract": "Background: Aflatoxin B1 (AFB1), a highly toxic mycotoxin, is one of the contaminants of food items such as corn, rice, nuts, and flour. This study aimed to evaluate the effect of AFB1 on the histology and ultrastructure of the submandibular salivary glands (SMSG) of albino rats and examine the possible therapeutic effect of Rosmarinus officinalis extract. Methods: This study used 21 adult male albino rats equally divided into three groups as follows: Group C (saline-treated control group); Group A (AFB1 treated group) subjected to intraperitoneal injection of AFB1 (2 mg/kg) once daily for four weeks; Group R (rosemary-treated group) subjected to AFB1 as in Group A followed by two weeks of intraperitoneal injection of Rosmarinus officinalis extract (400mg/kg) once daily. At the end of the experimental periods, SMSGs were excised and fixed for histological and ultrastructural examinations. Results: SMSGs of the AFB1 group presented atrophied serous acini with numerous cytoplasmic vacuolations; their granular convoluted tubules, striated ducts and excretory ducts presented signs of degeneration in their cell lining with the presence of abundant cytoplasmic vacuolations. In addition, dilated blood vessels engorged with red blood cells were frequently seen. Ultrastructural findings of the AFB1 group showed some acinar cells with degenerated mitochondria presenting loss of cristae and vacuolations as well as irregular, shrunken nuclei with condensed chromatin. Dilated rough endoplasmic reticulum were observed in granular convoluted tubules and striated ducts. The glands of animals that received rosemary extract almost regained their normal architecture. Conclusions: It can be concluded that rosemary extract has an ameliorative effect on the deleterious histological and ultrastructural changes induced by chronic AFB1 intake in rat SMSGs.", "keywords": [ "Aflatoxin B1", "Rosemary", "Submandibular salivary gland", "Antioxidant", "electron microscope" ], "content": "Introduction\n\nAflatoxins are naturally-occurring mycotoxins produced by the fungi Aspergillus flavus, a mold that can be found on several food products. They are probably the most popular and most intensively studied mycotoxins in the world1. Aflatoxins could contaminate major agricultural commodities like corn, wheat, rice, barley, maize, peanut, onions and many other crops. They are also found in the milk, eggs and meat of animals that feed on aflatoxin-contaminated food2,3. Aflatoxins are highly toxic and could cause both acute and chronic toxicity in human beings and animals4.\n\nOf the 20 types, Aflatoxin B1 (AFB1) is considered to be the most toxic. AFB1 toxicity was reported to lead to severe health issues such as cancer, growth retardation, and immunosuppression. In this regard, the International Agency for Research on Cancer classified AFB1 as a group I carcinogen, which raises the risk of cancer in humans5. The oxidative stress caused by AFB1 is considered the main mechanism of action for AFB1-induced cell DNA, protein and lipid damage. Many studies suggested that toxicity may occur from the production of reactive oxygen species (ROS) intracellularly during AFB1 metabolism in the liver6,7.\n\nAntioxidants act as ‘free radical scavengers’, which avert and repair the damage caused by ROS. They stabilize or deactivate free radicals before the latter attack cells and biological targets8. The scientific interest in the protective effect of antioxidants against aflatoxins has markedly increased in the last years9. Among the plants recognized to present a rich source of antioxidants is Rosmarinus officinalis (common name, rosemary). Rosemary extract has also been shown to inhibit the fungal growth of Asperigillus parasiticus and subsequent aflatoxin production10. In several studies rosemary has shown positive biological actions in terms of antioxidant activity, which reduced the harmful effects of AFB111–13.\n\nInformation on the effect of AFB1 on the SMSG structure has been insufficient. Therefore, the aim of the present study was to examine the effect of AFB1 on the SMSG of albino rats using light and electron microscopes as well as explore the potential therapeutic effect of rosemary extract on aflatoxin-induced changes. Rats were used in this study because they have biology similar to humans and therefore can be a model for human toxicity.\n\n\nMethods\n\nThe study protocol and the procedures for animal care and experiments were approved by the Research Ethics Committee of the Faculty of Dentistry, Ain-Shams University, Egypt (#615).\n\nAll efforts were made to ameliorate any suffering of the animals by adopting the OECD 423 test guidelines.\n\nThis study used 21 adult male albino rats, three months old and weighing 200-220 g. The animals were obtained from and housed in the Animal house, Faculty of Medicine, Ain Shams University. Sample size calculation was performed using GPower version 3.1.9.2 (University Kiel, Germany)14. The effect size was 0.95 using α level of 0.05 and β level of 0.05, i.e., power = 95%; the estimated sample size (n) was a total of 21 samples for three groups.\n\nRats were housed separately in cages (Suzhou Suhang Technology Equipment Co., Ltd.) in a constant temperature (22–24°C) and light-controlled room on an alternating 12:12 hour light-dark cycle and had free access to food and water. The animals were fed a natural diet and water ad libitum throughout the whole experiment.\n\nAll 21 rats were given a number (1–21) using a marker pen, then randomized by putting the numbers in an envelope and dividing them into three groups according to the numbers which were taken from the envelope\n\nThe three groups were as follows :\n\nGroup C (saline-treated control group): rats were injected with 9% saline intraperitoneally once daily for four weeks.\n\nGroup A (AFB1 treated group): rats were injected with AFB1 (Sigma Chemical Co., St. Louis, MO, USA) intraperitoneally with a dose of 2 mg/kg once daily for four weeks15.\n\nGroup R (rosemary treated group): rats were injected with AFB1 intraperitoneally using the same dose and period as in Group A followed by intraperitoneal injection of Rosmarinus officinalis extract (400mg/kg) for two weeks16.\n\nThe rats were injected every morning at 9 am in the animal house laboratory of Ain Shams university.\n\nDried rosemary leaves were obtained from Harraz for food industry, natural products and botanical herbs (Product number 215). Dried rosemary leaves were ground for methanolic extraction, which was done as described by Hendel et al.17. The obtained rosemary leaves were powdered and 100 g were submitted to hydrodistillation for 3 h with 1000ml distilled water using Clevenger type apparatus. The extracted oil was collected and dried over anhydrous sodium sulfate, then stored in sealed glass vials in a refrigerator at 4°C until use. From the plant extract material, 30 g was soaked for eight hours in methanol solvent at 40°C in a sterile conical flask. The mixture was then filtered using Whatman No. 4 filter paper to yield a light-brown filtrate. The methanolic filtrate was concentrated using a vacuum rotary evaporator (Heidolph 36001270 Hei-Vap Precision Rotary Evaporator) at 100 rpm for 30 minutes. The remaining extract was then dried in a vacuum oven for two hours to ensure the elimination of any residual solvent. The final extract was a dark green powder, which was then dissolved in 9% saline.\n\nAt the end of each experimental period, rats were euthanized by CO2 asphyxiation followed by cervical dislocation and their SMSGs were cut out performed by experienced animal laboratory personnal. SMSGs of the right side were fixed in neutral buffered formalin (10%), embedded in paraffin and sections 6 microns thick were sliced and stained with hematoxylin and eosin (H&E) for light microscopic examination using a Leica DM 1000 light microscope and camera and Leica Application suite-LAS software in the research center, The British University in Egypt. The specimens of the left sides were used for ultrastructural examination where small specimens were cut and fixed in 3% glutaraldehyde and osmium tetraoxide, dehydrated in alcohol and embedded in an epoxy resin. Microtome sections were prepared at approximately 500–1000 um thickness with a Leica Ultracut UCT ultramicrotome. Thin sections were stained with toluidine blue (1X), then sections were examined using a Lica ICC50 HD camera. Ultra-thin sections were prepared at approximately 75–90 um thickness and were stained with uranyl acetate and lead citrate, then examined using a JEOL (JEM-1400 TEM) transmission electron microscope.\n\n\nResults\n\nHistological examination of SMSG of the control group is shown in Figure 1A and Figure 1B, which revealed normal histological features of the submandibular salivary gland.\n\n(A, B) Control group showing regular gland architecture of serous acini (S) intercalated duct (circle), granular convoluted tubule (GCT), striated duct (ST) and excretory duct (EX) surrounded by fibrous connective tissue stroma (arrows) (H&E orig. mag x 400). (C, D) AFB1 treated group showing remnants of degenerated acini (red arrows), atrophic serous acini (S) with cytoplasmic vacuolation (black arrows), wide interacinar spaces (yellow arrows), vacuolated GCT and striated duct (ST) and excretory duct (EX) with thickened lining epithelium presenting extensive vacuolations (yellow arrows) and some crescent shaped nuclei in its cells (red arrows). Degenerated areas are seen in the surrounding fibrous connective tissue stroma (black arrows) (H&E orig. mag x 400). (E, F) Rosemary treated group showing normal serous acini (S), intercalated ducts (circle), GCT with few cytoplasmic vacuoles (arrows) and striated ducts with normal cell lining (ST). Dilated and congested blood vessels were observed (B) and excretory duct (EX) shows relatively normal lining with slight vacuolations in some areas (arrow) (H&E orig. mag x 400).\n\nHistological sections of SMSG of the AFB1 administered group rats showed acini apparently decreased in size, as revealed by wide interacinar spaces, and numerous different sized cytoplasmic vacuoles were seen in their cells (Figure 1C). Striated ducts showed a decrease in size and loss of basal striations in the lining cells was observed. The presence of abundant cytoplasmic vacuolations were frequently observed in both striated and granular convoluted tubule (GCT) cells. Excretory duct cells showed extensive intracytoplasmic vacuolations and crescent-shaped nuclei. Retained secretory material was frequently found inside its lumen (Figure 1D). Degeneration in the surrounding connective tissue fibers was also noted (Figure 1D).\n\nHistological examination of the SMSGs of the rosemary group rats presented almost identical histological features to the control group except with a few degenerative changes in both the acini and the ducts in some areas where few small intra-cytoplasmic vacuoles were detected. A limited number of congested and dilated blood vessels were observed close to striated ducts (Figure 1D). Some lining cells of the excretory duct exhibited vacuolations in certain areas (Figure 1F).\n\nControl group. Ultrastructural examination of the SMSG of group A rats showed normal serous acini that were spherical in shape containing basally situated rounded nuclei (open-faced nucleus) and a narrow central lumen (Figure 2A). Acinar cells exhibited parallel arrays of rough endoplasmic reticulum (rER) (Figure 2B) and oval-shaped mitochondria (Figure 2C). Secretory granules of variable sizes and different electron densities occupied most of the cytoplasm (Figure 2A). The nuclei of the serous cells appeared euchromatic with a smooth, regular nuclear membrane (Figure 2B). The GCTs presented long columnar cell lining. Their nuclei were large, rounded and basally located. Their cells contained numerous well-circumscribed membrane-bounded electron-dense secretory granules (Figure 2D). The intralobular striated ducts consisted of long columnar cells. The basal cell membrane of the lining cells showed complex extensive infoldings. Few vertically arranged mitochondria were found interspersed with the folded membranes (Figure 2E). The excretory duct was lined by pseudostratified epithelium containing open face nuclei (Figure 2F).\n\nElectromicrographs of the control group showing (A) acinar cells surrounding acinar lumen (L), rounded basally situated nuclei (N) and many secretory granules (SG) (uranyl acetate and lead x 5000). (B) Part of an acinar open faced nucleus (N) with regular nuclear membrane (arrow) and parallel arrays of rough endoplasmic reticulum (rER) (uranyl acetate and lead x 12000). (C) Normal acinar mitochondria with cristae (uranyl acetate and lead x 12000). (D) Granular convoluted tubule with columnar cells and numerous electron dense secretory granules (SG) and basally situated open faced nucleus (N) (uranyl acetate and lead x 2500). (E) Striated duct cells with open face nucleus (N), numerous basal infoldings (arrows) and radially arranged mitochondria (M) (uranyl acetate and lead x 5000). (F) Excretory duct showing pseudostratified epithelium containing open face nucleus (N) (uranyl acetate and lead x 2500).\n\nAFB1 group. Numerous large vacuolations were observed in the cytoplasm of acinar cells of AFB1 administered rats (Figure 3A). Their nuclei showed irregular outline and clumping of chromatin material and exhibited differences in their shape and size, with variable degrees of shrinkage and pyknosis (Figure 3A, Figure 3B and Figure 3C). rER presented luminal dilatation, discontinuity and fragmentation (Figure 3C). Some acinar cells showed mitochondria with loss of cristae and the presence of vacuoles (Figure 3B). The GCTs showed areas of degeneration in the form of a decrease in the number of electron-dense granules, intracytoplasmic vacuolations and dilatation in the rER (Figure 3D). The irregular shaped cells of the striated duct revealed signs of degeneration displayed as shrunken nuclei and irregular nuclear outline. Degenerated organelles (especially dilated rER) and intra-cytoplasmic vacuolations were frequently encountered in the duct cells. Loss of basal infoldings on the striated ducts was also noticed (Figure 3E). The excretory duct cells showed cytoplasmic vacuolations and nuclei with an irregular outline (Figure 3F).\n\nElectromicrographs of the AFB1 group showing (A) serous acinus with different sized intracytoplasmic vacuolations (V) and shrunk nuclei (N) (uranyl acetate and lead x 5000). (B) Open face nucleus of serous acinar cell (N) with irregular nuclear membrane (white arrows). Radially arranged mitochondria (M) with dilated cristae and vacuoles. Degenerated cytoplasm (black arrows) can be noticed (uranyl acetate and lead x 12000). (C) Serous cell with irregular nuclear membrane (N) & dilated rough endoplasmic reticulum (rER) (uranyl acetate and lead x 5000). (D) Granular convoluted tubule cells with intracytoplasmic vacuolations (arrows) and dilated rER (uranyl acetate and lead x 4000) (E) Striated duct with indistinct cell outlines and numerous large intracytoplasmic vacuolations (V) pressing on the cell nucleus and loss of basal striations in some cells (uranyl acetate and lead x 5000). (F) Excretory duct cells showing cytoplasmic vacuolations (V) and nuclei (N) with irregular outline (uranyl acetate and lead x 2500).\n\nRosemary treated group. Electron microscopic examination of SMSG of rats after treatment with rosemary revealed that the majority of acinar cells almost regained their normal architecture (Figure 4A). Slightly dilated rER and very few cytoplasmic vacuolations were observed (Figure 4B). GCTs were lined by long columnar cells with basally situated nuclei and many electron-dense secretory granules (Figure 4C). Striated ducts regained their normal histological appearance, with the columnar cells having numerous basally located radially arranged rod-shaped mitochondria with minimal changes. Desmosomal junctions appeared in a normal pattern in between cells (Figure 4D and Figure 4E). The excretory duct was lined by pseudostratified epithelium containing open face nuclei (Figure 4F).\n\nElectromicrographs of the rosemary treated group showing (A) serous acinus containing nucleus (N), numerous secretory granules and few cytoplasmic vacuoles (uranyl acetate and lead x 5000). (B) Open face nucleus (N) of an acinar cell and parallel arrays of rough endoplasmic reticulum (rER) with slight dilatations in some areas. Slight perinuclear space can be noticed (arrow) (uranyl acetate and lead x 12000). (C) Granular convoluted tubule (GCT) containing basally situated nuclei (N) and numerous electron dense secretory granules (SG) (uranyl acetate and lead x 2500). (D) Striated duct cell with open face nucleus (N) and tubular shaped mitochondria (M). Minimal cytoplasmic degeneration and some loss of basal infoldings can be observed (black arrows) (uranyl acetate and lead x 12000) (E) Striated duct columnar cells with basal infoldings (black arrows) and numerous rod shaped mitochondria (white arrows) (uranyl acetate and lead x 5000). (F) Excretory duct with pseudostratified epithelium lining cells presenting open face nucleus (N) (uranyl acetate and lead x 2500).\n\nOriginal microscopy light and electron microscopy images for all groups are provided as Underlying data18.\n\n\nDiscussion\n\nIn poor developing countries, chronic intake of foods contaminated with aflatoxins is a prevailing problem in both humans and animals worldwide. Aflatoxins are considered of great global concern due to their carcinogenic and toxic effects on human and animal health4. In the present study, AFB1 was administrated to the experimental rats for four weeks to simulate enough chronic exposure to cause oxidative stress in vital organs19. The rats were administered the toxin intraperitoneally to ensure the delivery of the full dose to each animal.\n\nBased on its popular use as an antioxidant in several studies on various tissues13,20, rosemary was chosen in this study to evaluate its potential for alleviating the histological and ultrastructural changes in the SMSG of aflatoxicated rats. Moreover, rosemary extracts have been found to have a positive effect on the natural antioxidants produced in the body. According to De Oliveira et al.11, rosemary improves the production of endogenous antioxidants, leading to enhancement of the body’s antioxidant capacity, reduction in the levels of lipid peroxidation, and maintenance of cell membrane permeability. Furthermore, rosemary was chosen in the current study rather than synthetic antioxidants based on the study by Mira-Sánchez et al.21, who found that the effect of rosemary compounds as an antioxidant was greater than the effect reported by artificial antioxidants.\n\nIn the present study, rosemary extract was administered at 400 mg/kg once daily for 14 days as Rahbardar et al.16 documented that treatment of albino rats with rosemary extract with this dose and duration was enough to elicit its therapeutic potential.\n\nThe present results showed that AFB1 administration caused degenerative histopathological alterations in the SMSG of albino rats in the form of shrunken acini and numerous cytoplasmic vacuolations in acini, GCTs and excretory ducts. These results were in agreement with the study of Abou-Zeid et al.22, who reported that SMSGs of rats exposed to AFB1 showed severe vacuolar degeneration in acinar cells and the epithelial cells of convoluted ducts. Moreover, the damaging effects of AFB1 detected in this study were also in parallel with other histopathological studies in which aflatoxins caused degeneration and numerous cytoplasmic vacuolations in hepatocytes of the liver and Langerhans cells of the pancreas of rats. The investigators attributed this to ROS production from the ingested aflatoxins23,24.\n\nCoppes et al.25 attributed the presence of acinar intracytoplasmic vacuoles to intra-cytoplasmic degenerative changes caused by the presence of proteolytic enzymes in their secretory granules which might infiltrate and cause damage to the cytoplasm, leading to autolysis and cellular death. However, Samah et al.26 suggested that cytoplasmic vacuolations seen in acinar, GCT and excretory duct cells might be due to the ROS formation, resulting in lipid peroxidation and damage of cell membranes as well as membranes of the cell organelles, ending in impairment of the energy-dependent Na+ K+ ion pumps in the cell. These changes lead to accumulation of Na+ inside the cells with entry of water resulting in cellular swelling and vacuolations. In the present study, histological examination of the striated and excretory ducts showed dilatation of their lumens with retained secretion following AFB1 administration. Mahmoud et al.27 attributed this finding to the accumulation of the salivary secretion and disturbance of exocytosis caused by glandular dysfunction. The changes in the connective septa reported in this study were inconsistent with several studies reporting that exposure to excessive amounts of toxins caused fibrosis and subsequently, overproduction of mature collagen fibrils22,28.\n\nIn this study AFB1 induced many alterations in SMSG tissue when observed under the electron microscope. AFB1 caused many signs of degeneration in the nuclei of the acinar cells manifested as an irregular outline and clumping of chromatin material in addition to changes in shape and size. In a study conducted to explore the apoptotic mechanism of AFB1, Mughal et al.29 attributed shrunken nuclei and dense chromatin to increased expression of many proapoptotic proteins. Subsequently, DNA fragmentation and chromatin condensation results.\n\nYasuno et al.30 suggested that the presented luminal dilatation of rER following AFB1 administration could be attributed to impaired secretory mechanisms and subsequently, the proteinaceous secretory material becomes concentrated within the arrays of the rER, which leads to their dilatation. Moreover, the degenerated mitochondria in the cells of the acini and ducts in the aflatoxin-administered group in this study might be due to ROS production and/or to the preferential binding of aflatoxins to mitochondrial DNA, which hinders ATP production, causing disruption of mitochondrial functions and mitochondrial membranes31.\n\nThis research revealed that rosemary extract ameliorated the damaging effects of AFB1 on SMSG, which was observed histologically and ultrastructurally, where most alterations caused by AFB1 administration were greatly reduced. The present results might be explained by Ghoneim & Arafat32 who reported that treatment with rosemary extract prevented oxidative stress caused by ROS on rat parotid salivary glands. They attributed this improvement to the antioxidative properties of rosemary, which resulted in the subsidence of oxidative stress. Moreover, De Oliveira et al.33 attributed the improvement in the mitochondrial structure observed upon treatment with rosemary to the effect of carnosic acid, which avoided mitochondrial membrane potential disturbance and reduced the levels of oxidative stress markers in mitochondrial membranes.\n\nFrom the present study, it could be concluded that chronic intake of AFB1 gave rise to deleterious histological and ultrastructural changes in the SMSGs of albino rats and that treatment with rosemary extract was able to ameliorate this effect.\n\n\nData availability\n\nFigshare: Effects of aflatoxin B1 on the submandibular salivary gland of albino rats and possible therapeutic potential of Rosmarinus officinalis: a light and electron microscopic study. https://doi.org/10.6084/m9.figshare.12644855.v218\n\nThis project contains the following underlying data:\n\n- Original, unedited electron microscopy images in JPG format (em1.jpg - em18.jpg)\n\n- Original, unedited light microscopy images in TIF format (lm1.tif - lm6.tif)\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\nHamid AS, Tesfamariam IG, Zhang Y, et al.: Aflatoxin B1-induced hepatocellular carcinoma in developing countries: Geographical distribution, mechanism of action and prevention. Oncol Lett. 2013; 5(4): 1087–1092. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEaton DL, Groopman JD: The toxicology of aflatoxins: human health, veterinary, and agricultural significance. Elsevier. 2013\n\nSzigeti G, Baranyi N, Kocsubé S, et al.: Role of Aspergillus species in mycotoxin contamination of agricultural products in Central Europe. 2012. Reference Source\n\nEnyiukwu D, Awurum A, Nwaneri J: Mycotoxins in stored agricultural products: Implications to food safety and health and prospects of plant-derived pesticides as novel approach to their management. Greener Journal of Microbiology and Antimicrobials. 2014; 2(3): 32–48. Publisher Full Text\n\nYilmaz S, Kaya E, Kisacam MA: The effect on oxidative stress of aflatoxin and protective effect of lycopene on aflatoxin damage. Aflatoxin-Control, Analysis, Detection and Health Risks. 2017; 67–90. Publisher Full Text\n\nBerg D, Youdim MBH, Riederer P: Redox imbalance. Cell Tissue Res. 2004; 318(1): 201–213. PubMed Abstract \| Publisher Full Text\n\nSohn DH, Kim YC, Oh SH, et al.: Hepatoprotective and free radical scavenging effects of Nelumbo nucifera. Phytomedicine. 2003; 10(2–3): 165–169. PubMed Abstract \| Publisher Full Text\n\nTowner RA, Qian SY, Kadiiska MB, et al.: In vivo identification of aflatoxin-induced free radicals in rat bile. Free Radic Biol Med. 2003; 35(10): 1330–1340. PubMed Abstract \| Publisher Full Text\n\nFaizal P, Satheeshan, B, Adarsh A, et al.: Antioxidant status and oxidative stress in the circulation of younger and elderly human subjects. Indian J Clin Biochem. 2013; 28(4): 426–428. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDa Silva Bomfim N, Kohiyama CY, Nakasugi LP, et al.: Antifungal and antiaflatoxigenic activity of rosemary essential oil (Rosmarinus officinalis L.) against Aspergillus flavus. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2020; 37(1): 153–161. PubMed Abstract \| Publisher Full Text\n\nDe Oliveira JR, Camargo SEA, De Oliveira LD: Rosmarinus officinalis L.(rosemary) as therapeutic and prophylactic agent. J Biomed Sci. 2019; 26(1): 5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHamidpour R, Hamidpour S, Elias G: Rosmarinus officinalis (Rosemary): a novel therapeutic agent for antioxidant, antimicrobial, anticancer, antidiabetic, antidepressant, neuroprotective, anti-inflammatory, and anti-obesity treatment. Biomed J Sci Tech Res. 2017; 1(4): 1–6.\n\nNaiel MA, Ismael NE, Shehata SA: Ameliorative effect of diets supplemented with rosemary (Rosmarinus officinalis) on aflatoxin B1 toxicity in terms of the performance, liver histopathology, immunity and antioxidant activity of Nile Tilapia (Oreochromis niloticus). Aquaculture. 2019; 511: 734264. Publisher Full Text\n\nFaul F, Erdfelder E, Buchner A, et al.: Statistical power analyses using GPower 3.1: Tests for correlation and regression analyses. Behav Res Methods. 2009; 41(4): 1149–1160. PubMed Abstract \| Publisher Full Text\n\nSakr S, Bayomy M, El-Morsy A: Rosemary extract ameliorates cadmium-induced histological changes and oxidative damage in the liver of albino rat. J Basic Appl Zool. 2015; 71: 1–9. Publisher Full Text\n\nRahbardar MG, Amin B, Mehri S, et al.: Anti-inflammatory effects of ethanolic extract of Rosmarinus officinalis L. and rosmarinic acid in a rat model of neuropathic pain. Biomed Pharmacother. 2017; 86: 1441–449. PubMed Abstract \| Publisher Full Text\n\nHendel N, Larous L, Belbey L: Antioxidant activity of rosemary (Rosmarinus officinalis L.) and its in vitro inhibitory effect on Penicillium digitatum. Int Food Res J. 2016; 23(4): 1725–1732. Reference Source\n\nShamel M: Effects of aflatoxin B1 on the submandibular salivary gland of albino rats and possible therapeutic potential of Rosmarinus officinalis: a light and electron microscopic study. figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12644855.v2\n\nZhao J, Shirley RB, Dibner JD, et al.: Comparison of hydrated sodium calcium aluminosilicate and yeast cell wall on counteracting aflatoxicosis in broiler chicks. Poult Sci. 2010; 89(10): 2147–2156. PubMed Abstract \| Publisher Full Text\n\nAhmad S, Hafez A, Hassan M, et al.: Influence of rosemary extract on immune responses and oxidative stress in mice intoxicated by aflatoxins. Nat Sci. 2011; 9(10): 54–63. Reference Source\n\nAzab A, Fetouh FA, Albasha MO: Nephro-protective effects of curcumin, rosemary and propolis against gentamicin induced toxicity in guinea pigs: Morphological and biochemical study. American Journal of Clinical and Experimental Medicine. 2014; 2(2): 28–35. Publisher Full Text\n\nManafi M, Hedayati M, Yari M: Effectiveness of rosemary (Rosmarinus officinalis L.) essence on performance and immune parameters of broilers during aflatoxicosis. Adv Life Sci. 2014; 4(3): 166–173. Reference Source\n\nAbou-Zeid A, Hassan N, Abou El-Yazeed M: Effect of aflatoxin contaminated diet on major salivary glands and the protective role of Ozone application. Life Sci Alliance. 2014; 11(8): 450–460. Reference Source\n\nAbdel-Latif MS, Elmeleigy KM, Aly TA, et al.: Pathological and biochemical evaluation of coumarin and chlorophyllin against aflatoxicosis in rat. Exp Toxicol Pathol. 2017; 69(5): 285–291. PubMed Abstract \| Publisher Full Text\n\nEl-Haleem MRA, Mohamed DA: The effects of experimental aflatoxicosis on the pancreas of adult male albino rats and the role of ginger supplementation: a histological and biochemical study. Egyptian Journal of Histology. 2011; 34(3): 423–435. Publisher Full Text\n\nEl-Sayed SK, Abo El-Yazed AA, El-Bakary NA, et al.: Histological and Immunohistochemical Study of the Effect of Alendronate on the Submandibular Salivary Gland of Adult Male Albino Rat and the Possible Protective Effect of Propolis. Med J Cairo Univ. 2018; 86: 3119–3132. Publisher Full Text\n\nAbdel-Wahhab MA, Hassan NS, El-Kady AA, et al.: Red ginseng extract protects against aflatoxin B1 and fumonisins-induced hepatic pre-cancerous lesions in rats. Food Chem Toxicol. 2010; 48(2): 733–742. PubMed Abstract \| Publisher Full Text\n\nShamel M, Al Ankily MM, Bakr MM: Epidermal growth factor restores normal levels of myosin expression in submandibular salivary glands of rats treated with botulinum toxin. J Adv Med Dent Scie Res. 2017; 5(1): 1–6. Reference Source\n\nMughal MJ, Peng X, Zhou Y, et al.: Aflatoxin B1 invokes apoptosis via death receptor pathway in hepatocytes. Oncotarget. 2017; 8(5): 8239–8249. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYasuno K, Igura S, Yamaguchi Y, et al.: Pathological examination of spontaneous vacuolation of pancreatic acinar cells in mice. J Toxicol Pathol. 2019; 32(2): 105–109. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu Y, Wang W: Aflatoxin B1 impairs mitochondrial functions, activates ROS generation, induces apoptosis and involves Nrf2 signal pathway in primary broiler hepatocytes. Anim Sci J. 2016; 87(12): 1490–1500. PubMed Abstract \| Publisher Full Text\n\nGhoneim FM, Arafat EA: Histological and histochemical study of the protective role of rosemary extract against harmful effect of cell phone electromagnetic radiation on the parotid glands. Acta Histochem. 2016; 118(5): 478–485. PubMed Abstract \| Publisher Full Text\n\nDe Oliveira MR, Peres A, Ferreira GC, et al.: Carnosic Acid Affords Mitochondrial Protection in Chlorpyrifos-Treated Sh-Sy5y Cells. Neurotox Res. 2016; 30(3): 367–379. PubMed Abstract \| Publisher Full Text" }	[ { "id": "67572", "date": "22 Jul 2020", "name": "Mahmoud Serag", "expertise": [ "Reviewer Expertise Dentistry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe present article by Bassant et al. is well written and the results are well presented in the manuscript. The methods were clearly described and thorough details were provided which adds to the scientific value of the manuscript. Power analysis details are provided for the sample size calculation. Light microscopic and electron microscopic photos are clear and described sufficiently.\nHowever, I have some remarks that might add to the manuscript:\nThe authors could add the use of an immunohistochemical marker for oxidative stress to confirm histological findings.\n\nGrouping could be renamed I, II and III instead of the current namings.\n\nIn the first group, normal saline is 0.9% not 9.\n\nI believe this manuscript is suitable for publication.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "67567", "date": "28 Jul 2020", "name": "Dina Rady", "expertise": [ "Reviewer Expertise Oral 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 idea of the research article titled: “Effects of aflatoxin B1 on the submandibular salivary gland of albino rats and possible therapeutic potential of Rosmarinus officinalis: a light and electron microscopic study” is very interesting and valuable as the researcher illustrated the effect of AFB1 on the submandibular salivary glands of albino rats. Besides, the authors investigated the possible therapeutic effect of Rosmarinus officinalis extract which could be a base for clinical application.\nThe article is clear and easy to comprehend as it is well constructed. Significant results were noted in the results of this study, all the photomicrographs are very clear. But still, there are suggested minor comments the authors could deal with, or at least discuss for additional impact.\n\nIn the methodology:\nThe letters used to identify different groups can be changed into numbers to be easily identified.\n\nIt is an idea to perform an additional assessment method. Example: RT-PCR analysis of α-amylase gene expression to confirm the obtained results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-752
https://f1000research.com/articles/9-751/v1	21 Jul 20	{ "type": "Data Note", "title": "The complete genome sequence of Stevia rebaudiana, the Sweetleaf", "authors": [ "Kathleen O'Neill", "Stacy Pirro", "Kathleen O'Neill" ], "abstract": "The Sweetleaf (Stevia rebaudiana: Asteraceae) is widely grown for use as a sweetener. We present the whole genome sequence and annotation of this species. A total of 146,838,888 paired-end reads consisting of 22.2G bases were obtained by sequencing one leaf from a commercially grown seedling. The reads were assembled by a de-novo method followed by alignment to related species.\n\nAnnotation was performed via GenMark-ES. The raw and assembled data is publicly available via GenBank: Sequence Read Archive (SRR6792730) and Assembly (GCA_009936405).", "keywords": [ "Stevia rebaudiana", "Sweetleaf", "genome", "assembly", "annotation" ], "content": "Introduction\n\nThe Sweetleaf (Stevia rebaudiana: Asteraceae) is cultivated commercially for use as a sweetener. The sweetness is due to various steviol glycosides, primarily stevioside and rebaudioside. These compounds have 200-300X the sweetness of sugar (Abdullateef & Osman, 2012) but have no calories. The market for raw Stevia and derived products is expected to exceed 1B USD by 2021 (International Stevia Council, 2017).\n\nStevia rebaudiana has been used as a sweetener for centuries in Brazil and Paraguay (Misra et al., 2011). Botanist Moisés Santiago Bertoni first described the plant as growing in eastern Paraguay and noted its use as a sweetener (Bertoni, 1899).\n\nChemists Bridel and Lavielle isolated the glycosides stevioside and rebaudioside that give the leaves their sweet taste (Bridel & Lavielle, 1931). The chemical structures of the aglycone steviol and its glycoside have been solved (Mosettig & Nes, 1955).\n\nA complete genome sequence for this species will assist with discovering markers for crop yields, disease and drought resistance, and determining the biochemical pathways for the relevant metabolites.\n\n\nMethods\n\nA single commercially grown Stevia rebaudiana plant was used for this study (Behnke Nurseries, Beltsville, MD, USA). DNA extraction was performed on tissue from a single leaf using the Qiagen DNAeasy genomic extraction kit for plants, using the standard process. A paired-end sequencing library was constructed using the Illumina TruSeq kit, according to the manufacturer’s instructions. The library was sequenced on an Illumina Hi-Seq platform in paired-end, 2 × 150bp format.\n\nThe resulting fastq files were trimmed of adapter/primer sequence and low-quality regions with Trimmomatic v0.33 (Bolger et al., 2014). The trimmed sequence was assembled by SPAdes v2.5 (Bankevich et al., 2012) followed by a finishing step using RagTag v1.0.0 (Alonge, 2020) to make additional contig joins based on conserved regions in related plant species: Erigeron canadensis (GCA_010389155), Mikania micrantha (GCA_009363875), and Helianthus annuus (GCA_002127325). Default parameters were used for all assembly steps.\n\nAnnotation was performed using GeneMark-ES v2.0 (Lomsadze et al., 2005). Annotation was performed fully de novo without a curated training set and default parameters.\n\n\nResults\n\nThe genome assembly yielded a total sequence length of 411,383,069 bp over 55,557 scaffolds with an N50 of 37,276,437. The GeneMark-ES annotation resulted in 24,994 genes.\n\n\nData availability\n\nRaw and assembled data is publicly available via GenBank:\n\nRaw genome of Stevia rebaudiana, Accession number SRR6792730: https://www.ncbi.nlm.nih.gov/sra/?term=SRR6792730\n\nAssembly of Stevia rebaudiana, Accession number ASM993640v1: https://www.ncbi.nlm.nih.gov/assembly/GCA_009936405.1/", "appendix": "References\n\nAbdullateef RA, Osman M: Studies on effects of pruning on vegetative traits in Stevia rebaudiana Bertoni (Compositae). Int J Biol. 2012; 4(1). Publisher Full Text\n\nAlonge M: Ragtag: Reference-guided genome assembly correction and scaffolding. GitHub archive. 2020. Publisher Full Text\n\nBankevich A, Nurk S, Antipov D, et al.: SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing. J Comput Biol. 2012; 19(5): 455–477. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBertoni MS: Revista de Agronomia de l'Assomption. 1899.\n\nBolger AM, Lohse M, Usadel B: Trimmomatic: A flexible trimmer for Illumina Sequence Data. Bioinformatics. 2014; 30(15): 2114–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBridel M, Lavielle R: Sur le principe sucre des feuilles de kaa-he-e (stevia rebaundiana B). Comptes rendus de l'Académie des Sciences. 1931; 192: 1123–5.\n\nInternational Stevia Council: Industry data report, September 2017. 2017.\n\nLomsadze A, Ter-Hovhannisyan V, Chernoff YO, et al.: Gene identification in novel eukaryotic genomes by self-training algorithm. Nucleic Acids Res. 2005; 33(20): 6494–6506. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMisra H, Soni M, Silawat N, et al.: Antidiabetic activity of medium-polar extract from the leaves of Stevia rebaudiana Bert. (Bertoni) on alloxan-induced diabetic rats. J Pharm Bioallied Sci. 2011; 3(2): 242–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMosettig E, Nes WR: Stevioside. II. The structure of the aglucon. J Org Chem. 1955; 20(7): 884–899. Publisher Full Text" }	[ { "id": "68815", "date": "07 Aug 2020", "name": "Eric Lu Zhang", "expertise": [ "Reviewer Expertise Computational genomics", "bioinformatics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors sequenced and assembled the genome of Sweetleaf by Illumina short-reads and annotated the genes using de novo prediction. The data could be useful to the filed, but some points need to be clarified:\nCommonly, the first step of genome assembly is to estimate the genome size using k-mer frequency distribution. The authors should add such analysis in the revision.\n\nBased on the results in (1), the genome size of Stevia rebaudiana is ca. 1.3Gb. I don't think SPAdes is a good choice as it only works well on the small geneomes.\n\nGene prediction is too simple and may loose considerable genes. The prediction should include multifaceted information, such as de novo prediction, Homology-based genes prediction et al.\n\nAdd a citation for \"standard process\" of DNA extraction and the model of sequencing platform, e.g. Hi-Seq 2500.\n\nIt is better to include a table to show the statistics, such as N50, genome size, the number of genes.\n\nBoth of the assemblies from short-reads and short-reads+RagTag should be provided, becasue RagTag may introduce certain bias.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "67544", "date": "07 Aug 2020", "name": "Andrew Miller", "expertise": [ "Reviewer Expertise Sanger sequencing", "mycology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 the whole genome sequence of Sweetleaf, the plant where the sweetener, Stevia, is derived. I am surprised someone has not already sequenced this genome - it is certainly timely. The data are well presented and publicly available through NCBI. It is well-written and worthy of indexing. I only have minor comments below:\nShould the keywords repeat words found in the title?\n\nCan more be said in the Results about the genome?\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": "74315", "date": "18 Nov 2020", "name": "Dawson White", "expertise": [ "Reviewer Expertise Plant systematics." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nStevia is an economically important plant with no prior genomic resource for researchers. Standard methods were used for laboratory preparation, sequencing, assembly, and annotation of the genome. The resulting assembly and gene annotations are good based on the provided statistics.\nI would like to see that a voucher specimen of the sequenced plant (or a similar individual) at Behnke Nursuries was deposited in a herbarium. This is important for reproducible science and its omission almost causes me to mark that the methods are only \"partly\" sufficient.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-751
https://f1000research.com/articles/9-292/v1	24 Apr 20	{ "type": "Opinion Article", "title": "Turning up the heat on COVID-19: heat as a therapeutic intervention", "authors": [ "Marc Cohen" ], "abstract": "Enveloped viruses such as SAR-CoV-2 are sensitive to temperature and are destroyed by temperatures tolerable to humans. All mammals use fever to deal with infections and heat has been used throughout human history in the form of hot springs, saunas, hammams, steam-rooms, sweat-lodges, steam inhalations, hot mud and poultices to prevent and treat respiratory infections and enhance health and wellbeing. This paper reviews the evidence for using heat to treat and prevent viral infections and discusses potential cellular, physiological and psychological mechanisms of action. In the initial phase of infection, heat applied to the upper airways can support the immune system’s first line of defence by supporting muco-ciliary clearance and inhibiting or deactivating virions in the place where they first lodge. This may be further enhanced by the inhalation of steam containing essential oils with anti-viral, mucolytic and anxiolytic properties. Heat applied to the whole body can further support the immune system’s second line of defence by mimicking fever and activating innate and acquired immune defences and building physiological resilience. Heat-based treatments also offer psychological benefits by directing focus on positive action, enhancing relaxation and sleep, inducing 'forced-mindfulness', and invoking the power of positive thinking and remembered wellness. Heat is a cheap, convenient and widely accessible therapeutic modality and while no clinical protocols exist for using heat to treat COVID-19, protocols that draw from traditional practices and consider contraindications, adverse effects and infection control measures could be developed and implemented rapidly and inexpensively on a wide scale. While there are significant challenges in implementing heat-based therapies during the current pandemic, these therapies present an opportunity to integrate natural medicine, conventional medicine and traditional wellness practices, and support the wellbeing of both patients and medical staff, while building community resilience and reducing the likelihood and impact of future pandemics.", "keywords": [ "Heat stress", "hyperthermia", "sauna", "steam inhalation", "balneotherapy", "COVID-19" ], "content": "Heat in viruses and mammals\n\nLife exists within a narrowly defined temperature range, yet viruses, which are not technically alive, can remain biologically active in a wide range of environments. Enveloped viruses, such as rhinoviruses and coronaviruses, are most active in cool dry conditions, which are associated with increased occurrence of respiratory tract infections (Makinen et al., 2009), including infections with SARS-CoV (Chan et al., 2011) and SAR-CoV-2 (Sajadi et al., 2020; Wang et al., 2020). While enveloped viruses can remain active for long periods in cold conditions, their lipid envelopes are destroyed by temperatures tolerable to humans. The heat sensitivity of viruses is used routinely to deactivate viruses within vaccines, and temperatures of 55 to 65°C for 15 to 30 minutes are reported to deactivate a range of enveloped viruses, including coronaviruses (Darnell et al., 2004; Duan et al., 2003; Hu et al., 2011; Kampf et al., 2020; Lelie et al., 1987; WHO Report, 2003).\n\nThe first line of defence against respiratory viruses is the nasal cavity and sinuses, which maintains a protective mucosal barrier that allows viruses to be trapped, identified by the immune system and then swept away, as well as serving an important thermoregulatory role. The upper airways are constantly exchanging heat with inhaled air through convection, conduction and evaporation, which serves to cool inhaled air in summer and warm and humidify air in winter (Soni & Nayak, 2019). The upper airways also filter inhaled air and trap foreign particles and pathogens in a layer of watery mucus that is continually moved by cilia towards the pharynx, where it is either swallowed or expelled by coughing, sneezing and nose blowing. A moist mobile mucosal barrier is vital in the defence against respiratory infections and this barrier is enhanced by warm humid conditions and impaired by cigarette smoke and particulate pollution (Fahy & Dickey, 2010).\n\nIn winter when sunlight is restricted and the air is cold and dry, the nasal cavity becomes the coldest part of the body and if the airways dry out and the mucous becomes thicker and more difficult to clear, conditions become more favourable for viral penetration and replication. The ability of cool, dry conditions to enhance viral infection has been demonstrated in mice, with humidity of around 20% being shown to slow muco-ciliary clearance, impair innate antiviral defence, and reduce tissue repair function, leading to more rapid and severe illness compared with humidity of 50% (Kudo et al., 2019).\n\nIf respiratory viruses get past the first line of defence, fever is produced as part of the acute phase response which forms the immune system’s second line of defence. Fever is a cardinal response to infection that has been conserved throughout vertebrates for more than 600 million years. Ectotherms as diverse as reptiles, fish, and insects raise their core temperature during infection through behavioural regulation, and all mammals have evolved sophisticated mechanisms to create and disperse heat and manage the oxidative stress associated with operating at higher temperatures (Evans et al., 2015).\n\n\nMechanisms of action\n\nThe mechanisms by which heat overcomes viral infections depends on the setting, source, temperature, humidity, location and time course of applied heat. Whether internally generated or externally applied, heat has a profound influence on host defences and physiological resilience, as well as on viral load and virulence, and engages adaptive thermoregulatory mechanisms that can increase or decrease body temperature in order to restore homeostasis (Schieber & Ayres, 2016).\n\nInhalation of hot air can support the immune system’s first line of defence by directly inhibiting or deactivating virions in the upper airways where they first lodge and supporting muco-ciliary clearance, which can be further enhanced by inhalation of steam (Gujrathi et al., 2016). Heat applied to the whole body further supports the immune system’s second line of defence by inducing heat-stress that mimics the effects of fever (Schieber & Ayres, 2016). Fever has multiple actions when dealing with infections that includ direct inhibition of pathogens, stimulation of both the innate and adaptive arms of the immune system and activation of regulatory processes that serve to dampen inflammatory responses and avoid excessive tissue damage during the return to thermal homeostasis (Evans et al., 2015).\n\nFebrile temperatures activate multiple cellular responses that include complex reciprocal regulation between immune system activation, inflammation and the heat shock response pathway (Singh & Hasday, 2013). While the mechanisms by which heat-stress modulates immune function are not fully understood, higher temperatures have been shown to activate immune cells by making their cell membranes more fluid, which increases cell differentiation and activation by viral antigens and enables a faster and more effective response to viral threats (Mace et al., 2011). Acute heat stress has also been shown to increase the TNF-alpha response of monocytes (Zellner et al., 2002), enhance interleukin-2 induced activity of Natural Killer (NK) cells (Kappel et al., 1991), and cause a 10-fold increase in interferon-γ production by T-lymphocytes (Downing et al., 1988). Regular heat-stress has also been shown to reduce adrenaline and cortisol, increase the cytotoxicity of NK cells, and enhance the proliferative response of B cells (Tomiyama et al., 2015). Heat-stress also stimulates the release of Heat Shock Proteins (HSPs), (Iguchi et al., 2012) which play an important role in antigen presentation and cross-presentation, activation of macrophages and lymphocytes, and activation and maturation of dendritic cells (Tsan & Gao, 2009) as well as serving a chaperone function and protecting immune cells and proteins from heat-induced damage (Singh & Hasday, 2013).\n\nIn addition to enhancing cellular responses, heat-stress increases cardiac output, plasma volume and peripheral blood flow, and induces detoxification through the liver and kidneys, as well as through the skin via sweating (Crinnion, 2011) through which some toxic elements are preferentially excreted (Genuis et al., 2011). Heat-stress also induces a hormetic stress response that builds physiological resilience and confers tolerance to subsequent stress in a similar way to exercise (Gálvez et al., 2018). The effects of heat stress may be further enhanced when it is followed by intermittent cold exposure, which shunts blood to internal organs and induces a diuresis (Epstein, 1978), and further aids in detoxification (Cochrane, 2004). Heat-stress also enhances the immunostimulatory effects of cold exposure on the innate immune system, which include leukocytosis, granulocytosis, an increase in NK cell count and activity, and an elevation in circulating levels of IL-6 (Brenner et al., 1999).\n\nHeat-stress may offer a further advantage against respiratory viral infections by altering blood pH. Hyperthermia induces hyperventilation and subsequent respiratory alkalosis (Tsuji et al., 2016), that creates alkaline conditions that may be more favourable to host defenses. The ability of a transient alkaline environment to inhibit viral replication and reduce infectivity has been demonstrated with human coronavirus 229E, which has maximal infectivity in acid conditions (Lamarre & Talbot, 1989), and with coronavirus MHV-A59, which undergoes conformational changes in the spike glycoprotein at a pH of 8 at 37°C degrees, which leads to rapid and irreversible inactivation and loss of infectivity (Sturman et al., 1990).\n\nIn addition to offering physiological advantages in the battle against viral infection such as COVID-19, heat also confers many psychological advantages. Sauna bathing and other forms of heat therapy require time and effort to be devoted towards active relaxation that can help divert attention from anxiety-producing news and/or relieve boredom associated with social confinement. Sauna bathing also enhances sleep (Hussain et al., 2019), which further supports immune function (Irwin & Opp, 2017). Engaging in an activity with an intended positive outcome can also impart feelings of control that may otherwise be lacking, and doing something that feels good and having positive expectations elicits the power of positive thought and the placebo effect or ‘remembered wellness’ (Benson & Friedman, 1996). Furthermore, the exploration of heat-tolerance induces a 'forced-mindfulness' and a focus on the breath, which has additional physical and psychological benefits (Black & Slavich, 2016). In a time of social distancing, saunas can also provide a way for close family members to come together in ways that have supported family cohesion for generations in Finland and other Nordic countries (Mather & Kaups, 1963).\n\n\nHeat as medicine\n\nThe use of heat for cleansing and healing forms a conscious extension of mammals’ use of heat that has been practiced throughout human history. Hot springs, saunas, hammams, steam rooms, sweat lodges, steam inhalations, baths, hot mud and poultices have been used traditionally in cold, dry climates to prevent and treat respiratory infections and to enhance overall health and wellbeing. While heat-based therapies are not widely used in mainstream medicine, other than the local application of hot packs for symptomatic relief, heat-based treatments are standard offerings in wellness establishments, such as hot springs, bathing facilities, gymnasiums, fitness centers, hotels and resorts, where they are used both therapy and recreation (Clark-Kennedy & Cohen, 2017).\n\nThere are multiple lines of evidence to support the use of heat and humidity for the prevention and treatment of viral respiratory infections. Historical and emerging evidence suggests regular sauna bathing enhances cardiovascular, respiratory and immune function as well as improving mood and quality of life (Hussain & Cohen, 2018). Epidemiological evidence further suggests that frequent sauna bathing is associated with a reduced risk of pneumonia and viral infection (Kunutsor et al., 2017), and randomised controlled trial evidence suggests that regular saunas can halve the incidence of respiratory viral infections (Ernst et al., 1990). Randomised controlled trials further suggest hot air can treat respiratory infection with humidified air at temperatures above 43°C for 20 to 30 minutes being found to reduce viral shedding, giving immediate relief of symptoms and improving the course of the common cold (Tyrrell, 1988; Tyrrell et al., 1989).\n\n\nClinical applications and implications\n\nThere are a range of heat-based interventions that can be used alongside social distancing, hand washing and other personal hygiene measures to aid in overcoming COVID-19. For example, warming and humidifying indoor environments can prevent drying of the nasal mucosa, increase muco-ciliary clearance and nasal patency, and provide symptomatic relief (Ophir & Elad, 1987). The direct application of heat to the upper airways at the first signs of infection may further serve to inhibit or deactivate virions in the place where they first lodge. This has been demonstrated in vitro with temperatures of 45°C for 20 minutes activating immune cells and releasing HSPs while suppressing rhinovirus multiplication by more than 90% (Conti et al., 1999). The inhalation of steam with added essential oils with anti-viral, decongestant, anxiolytic and other properties, may further assist in facilitating muco-ciliary clearance and reducing viral load as well as providing physical and psychological relief (Ali et al., 2015; Lee et al., 2017).\n\nInducing mild heat-stress through the use of hot springs (balneotherapy), hot baths, saunas, steam-rooms and application of hot mud (pelotherapy), can be used to mimic fever and activate immune defenses. Enhanced immunity has been demonstrated with hyperthermia induced by traditional Finnish saunas (Pilch et al., 2013), far-infrared saunas (Sobjima, 2018), heated nano-mist (Tomiyama et al., 2015), hot baths (Downing et al., 1988; Kappel et al., 1991; Tsuchiya et al., 2003; Zellner et al., 2002) and geothermal mineral water (Uzunoglu et al., 2017). The beneficial effects of heat-stress may be further potentiated by the traditional practice of alternating heat with exposure to cold, which has been shown to increase NK cell count and activity and raise circulating levels of IL-6 and norepinephrine (Brenner et al., 1999). This may translate into greater resistance to viral infections as evidenced by a randomised controlled trial that found regular hot and cold showers reduced work absenteeism during an influenza outbreak (Buijze et al., 2016).\n\nIn recent years far-infrared (FIR) saunas have been used as an alternative to the traditional Finnish saunas. These saunas use infrared emitters without water or humidity and generally run at lower temperatures than Finnish saunas. While the use of FIR saunas to treat viral infection has not been studied, FIR radiation is reported to deactivate single-strand RNA viruses (Huang & Li, 2020) and FIR saunas have been shown to raise body temperature and induce hormetic stress responses, which support host defenses (Shemilt et al., 2019).\n\nThere are currently no clinical protocols for using heat in the treatment of COVID-19, yet heat has a long history of traditional use, and traditional practices such as alternating hot and cold immersions, post-heat relaxation and use of essential oils can inform their development. Heat-based clinical protocols must consider temperature, timing and individual tolerance, along with humidity, as water is 25 times more conductive than air, making steam rooms tolerable at temperatures around 50°C while dry saunas can be tolerated at temperatures above 100°C. Clinical protocols are needed to design future studies and inform clinical practice and while sauna bathing is generally well tolerated, protocols must consider contra-indications such as unstable angina, severe infection, high fever, or concomitant alcohol consumption (Hannuksela & Ellahham, 2001) and the risk of adverse events, such as fainting, dizziness and burns, along with the risk of cross-infection.\n\nWhile the clinical application of heat has promise in the prevention and treatment of COVID-19, there are significant challenges in implementing heat-based therapies. The current pandemic has seen the fear of infection lead to the widespread closure of public facilities that offer saunas and heat treatments, such as bathing facilities, commercial hot springs, spas, gymnasiums, hotels and fitness centers, and while some countries such as Finland have a large number of private saunas, in most other locations private sauna ownership is limited to people with high socio-economic status. Thus, if sauna bathing is to be widely implemented, public bathing and sauna facilities will need to adopt infection control measures similar to those for dealing with COVID-19 in hospitals and medical facilities (Liang, 2020).\n\nHeat is one of oldest forms of microbial control and still remains one of the most common methods for controlling and eradicating pathogens. The temperatures achieved within a sauna are well within the range required for pathogen control and often exceed temperatures of 60°C for 30 min, 65°C for 15 min or 80°C for 1 min, which have been shown to reduce coronavirus infectivity by at least 4 log10 (Kampf et al., 2020). While the temperatures, humidity and times required to specifically deactivate SAR-CoV-2 in vivo are yet to be determined, the temperature within a sauna makes risk of cross infection in public facilities more likely to arise in changing rooms and ancillary spaces rather than within saunas themselves. Strategies for limiting cross infection in bathing facilities therefore need to include disinfection of public areas and managing social distancing and other behavior of staff and bathers.\n\nStrategies for limiting cross infection, including procedures for maintaining social distancing by limiting group size, have been recently developed for re-opening some of the 3000 hot springs that were recently closed in China (Wang, 2020). There are also moves to reopen facilities as quarantine zones where people can undergo heat treatments during isolation or as places of respite for overworked medical staff. It may also be possible for saunas and steam rooms to be included within hospitals and rehabilitation facilities for use by both patients and staff. Furthermore, simple home-based protocols can provide guidance on using heat for people who are currently self-isolated in their homes or in quarantine. Such protocols could be evaluated using crowd-sourced, citizen-science platforms, which may help to develop, test and optimize treatment strategies for current and future pandemics.\n\n\nConclusion\n\nHeat is a cheap, convenient and widely accessible therapeutic modality with a long history of traditional use, yet it remains to be seen whether heat can be effective in the treatment or prevention of COVID-19. The relatively low cost and wide availability of heat-based treatments, along with multiple mechanisms of action that include both physical and psychological dimensions, makes heat an attractive option for combating viral infections. The integration of these ancient forms of treatment with modern technology may lead to a greater integration of natural therapies in mainstream healthcare, with the potential to support the wellbeing of both patients and medical staff. This may also lead to a greater convergence between the healthcare and wellness industries, and the development of systems and activities that build the wellbeing and resilience of the wider community, thereby reducing the impact of future pandemics.\n\n\nData availability\n\nNo data is associated with this work.", "appendix": "References\n\nAli B, Al-Wabel NA, Shams S, et al.: Essential oils used in aromatherapy: A systemic review. Asian Pac J Trop Biomed. 2015; 5(8): 601–611. Publisher Full Text\n\nBenson H, Friedman R: Harnessing the power of the placebo effect and renaming it \"remembered wellness\". Annu Rev Med. 1996; 47: 193–199. PubMed Abstract \| Publisher Full Text\n\nBlack DS, Slavich GM: Mindfulness meditation and the immune system: a systematic review of randomized controlled trials. Ann N Y Acad Sci. 2016; 1373(1): 13–24. 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Repasky", "expertise": [ "Reviewer Expertise Immunology", "Thermal stress", "hyperthermia", "anti-tumor immunity", "Stress" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, interesting, and generally well-written article summarizing many of the positive benefits of thermal therapies, provided in the context of potential therapies for patients with COVID-19. In general, the authors cover a wide range of literature related to applications (local/regional, whole body, topical) of elevated temperature. My comments related only to organization and some references and distinguishing better the difference between fever and hyperthermia. Heat treatments are likely to be hyperthermia treatments. While fever contains a thermal element, fever and hyperthermia represent very different physiological circumstances.\n\nFor example, statements such as that in Paragraph 2, right column, page3 \"Fever has multiple actions with dealing with infections that include direct inhibition of pathogens, stimulation of both the innate and adaptive arms of the immune system and activation....\". While the authors provide a review article as a reference here, most of this literature deals with hyperthermia and not actual infectious fever and this difference needs to be highlighted better. One would not be treated with fever. Moreover, there are other fundamental differences. Febrile individuals are usually quite cold and surface vessels constricted to conserve heat and help raise body temperature. Hyperthermia causes sweating and surface blood vessel dilation because the set point is not elevated. Moreover, there is a massive literature on heat stress/stroke (from hyperthermia, not fever) that can kill people because of excessive adhesion of platelets and white blood cells in capillaries. While the authors do rightly mention some safety concerns, it is perhaps a bit insufficient with regard to the impact of both hyperthermia (and fever). At the same time, the scope of the beneficial effects of mild elevation of body temperature and local temperature (in particular with respect to viral infections) is quite important to remember and hopefully expand upon in future clinical applications.\nSome topics should be highlighted more. For example, there is a substantial literature using electromagnetic heating and infrared heating for cancer therapy and so there is already safe equipment available for both local/regional as well as surface heating. Reviews on this topic have been available for some time. This technology could already be available for COVID-19 patients.\n\nI found some of the referencing related to the direct impact of heat on viral replication (either in vivo (e.g., nasal cavities or in vitro) to be scattered a bit throughout the review. Some better reorganization of these various sections would help the reader to assess this very important point.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "5717", "date": "21 Jul 2020", "name": "Marc Cohen", "role": "Author Response", "response": "I agree that this is a timely and interesting topic that covers a wide range of literature and am grateful for your comments, which have led to improvements in the paper's content and flow. I have now modified the wording to better reflect the difference between fever and externally applied heat stress and have referenced a review of the different forms of heat treatment that are currently being used or are under investigation for cancer therapy and suggest they may be useful for treating COVID-19. I have also added more detail on the adverse effects of heat and have reorganised some sections to improve the flow of information and make the paper more cohesive." } ] }, { "id": "62786", "date": "07 Jul 2020", "name": "Jari Laukkanen", "expertise": [ "Reviewer Expertise Cardiovascular diseases", "sauna and exercise" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHeat is a cheap, convenient and widely accessible therapeutic modality. Sauna bathing is a form of heat therapy. However, there are no very accurate details of temperature, weekly frequency, and duration which would be needed to achieve those beneficial effects. An only traditional Finnish sauna is based on high temperatures.\nYou should discuss more with the lights of many published original studies on sauna and its protective health effects. The literature review of this narrative has missed many recent papers on this topic. Please consider adding the recently published key references on sauna and health outcomes so that this article would be more up-to-date.1,2,[ref-3,4,5,6,7\nWhat is the role of improved hygiene control due to regular sauna bathing?\nWarm and cold has any effect on the results?\nYou could add studies that may indicate that sauna is related to inflammatory markers or cytokines.\n\nIt is written that emerging evidence suggests regular sauna bathing enhances cardiovascular function. No relevant references were available in the manuscript.\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": "5718", "date": "21 Jul 2020", "name": "Marc Cohen", "role": "Author Response", "response": "Thank you for your suggestions. I have expanded the content on cardio-respiratory function and added all the suggested references on sauna bathing and health outcomes." } ] } ]	1	https://f1000research.com/articles/9-292
https://f1000research.com/articles/9-728/v1	20 Jul 20	{ "type": "Method Article", "title": "Modelling structural rearrangements in proteins using Euclidean distance matrices", "authors": [ "Aleix Lafita", "Alex Bateman" ], "abstract": "Proteins undergo large structural rearrangements such as circular permutations, dimerisation via domain swapping, and loss of core secondary structure elements in domain atrophy, among others. These structural changes can be naturally represented as distance matrix transformations, exploiting their conserved native residue contacts at the protein core. Here we present an homology modelling approach to formulate structural rearrangements as a Euclidean distance matrix (EDM) problem and use it to build their 3D structures. This modelling approach aims to be lightweight, flexible and fast, suitable for large-scale analyses. Models are typically coarse-grained and solely based on protein geometry. We demonstrate various applications of EDM-based modelling for protein structure analysis and release an open repository with the source code at: https://github.com/lafita/protein-edm-demo.", "keywords": [ "protein modelling", "structural rearrangements", "circular permutation", "domain swapping", "domain atrophy", "Euclidean distance matrices" ], "content": "Introduction\n\nMost proteins fold into compact globular domains stabilised by hydrogen bonds and a hydrophobic core. Protein domains typically allow for sequence mutations and short indels, which have a minor effect on the domain core. Some domains additionally allow for larger structural rearrangements that conserve the majority of core interactions. Some examples are domain swapping, where two independent domains exchange secondary structures to form an intertwined dimer1 circular permutations, where the sequence order of the domain is rearranged, connecting the N and C-termini and introducing new termini at a specific \"cut\" position2 and domain atrophy, where complete core secondary structures of a domain are deleted3.\n\nThese structural rearrangements have been extensively studied both experimentally and computationally due to their importance for protein stability and function4–6. A number of methods have been devised to predict domains susceptible to circular permutations and domain swaps7–9. However, modelling the 3D structure of the alternate conformations proves challenging, often requiring complicated software pipelines and long simulations8,10. We present an alternative modelling approach that consists in representing domains as Euclidean distance matrices (EDMs) and generating rearranged structures by applying a series of matrix operations (Figure 1).\n\nAtomic coordinates of the native SH3 domain (PDB:1SHG) structure (top left) are converted into a distance matrix (DM), shown at the bottom left as a heatmap of C-beta distances in a viridis color scale. A matrix transformation operation corresponding to the structural rearrangement is applied to the distance matrix (bottom middle), here shown as a red arrow for the circular permutation cut position and red boxes encapsulating conserved regions of the matrix. Values for unknown entries in the transformed distance matrix, shown as white stripes, are filled using an EDM completion algorithm to generate a complete EDM (bottom right). The distance matrix is finally converted to points in 3D corresponding to the atomic coordinates of the rearranged domain structure (top right).\n\nEDMs are matrices of squared distances between points in an m-dimensional Euclidean space11. By definition, they are square and symmetric matrices with zero diagonal and non-negative off-diagonal values, fulfilling the triangle inequality. Thanks to their many useful properties they have been used for applications in sensor localisation, molecular conformation and dimensionality reduction, among others. In proteins, EDM-based algorithms have been previously used to model structures from distance constraints derived from nuclear magnetic resonance (NMR) experiments12. EDMs are especially well suited for modelling protein structural rearrangements, since most of the distances at the domain core are conserved and can therefore be used as known entries in the matrix. Here we describe in detail a modelling approach based on EDMs and demonstrate its applications for studying circular permutations, domain swaps and domain atrophy in proteins.\n\n\nMethods\n\nGiven an input protein structure, its distance matrix can be computed as the Euclidean norm between all pairs of atoms. The distance matrix of a protein with n atoms will be n columns by n rows for a total of n2 entries. The granularity of the distance matrix can be easily adjusted by changing the number and type of atoms included from each protein residue. For example, a coarse-grained representation of a protein can be achieved using only C-alpha atoms.\n\nDistance matrices are invariant to point rotations, translations, and mirror images. This means that proteins rotated and translated in space have the same distance matrix. To the advantage of protein structure analysis, distance matrices of similar proteins can be directly compared without the need to superpose them in space. On the other hand, a protein and its non-natural mirror image are also indistinguishable from their distance matrix. Distance matrices can be inverted into a set of points in an embedding space of m dimensions using a technique known as multidimensional scaling (MDS). Distance matrix inversions can be challenging for noisy and incomplete matrices. Thanks to the degeneracy in low-rank protein distance matrices, where the number of points is much higher than the number of dimensions (three), MDS can handle small levels of noise and produce accurate reconstructions. Atomic coordinates of proteins can be generated from distance matrices using MDS in three dimensions. If the MDS point reconstruction results in a non-natural mirror image of a protein, its natural stereoisomer can be obtained by simply changing the sign of the z dimension of all points.\n\nPartial or incomplete EDMs, where only a subset of distances in the matrix are known, are a common problem in many applications. Finding values for the missing entries such that the resulting matrix is still an EDM is known as the EDM completion (EDMC) problem. Several different algorithms formulated as optimisation problems have been devised to complete EDMs, ranging from semidefinite programming (SDP) to numerical optimization, each with different loss functions and constraints.\n\nEDMC algorithms only converge to an exact solution with zero loss, in which the resulting matrix is an EDM. In some cases, there are multiple exact solutions to the completion problem and algorithms will randomly converge to one of the solutions. In other cases, convergence will not be possible and algorithms will return a solution with minimal loss instead, corresponding to the closest matrix to an EDM.\n\nFor the purpose of protein modelling, the dissimilarity parameterization formulation (DPF) algorithm13 offers a suitable solution. The dimensions of the EDM embedding space and upper and lower bounds for unknown distances are built into the loss function and can be set as input parameters. Thus, atomic distance bounds can be used to avoid clashes and unrealistic conformations.\n\nTo obtain realistic protein conformations in EDM models, we use atomic distances extracted from experimental structures in order to set upper and lower bounds on unknown entries in protein distance matrices. We selected one manual representative domain from each architecture type of the Evolutionary Classification of Protein Domains (ECOD) database14. The set of 20 domain structures is of diverse length, secondary structure content and fold topology, and determined by high-resolution X-ray crystallography (below 2Å).\n\nIn total, we calculated over three million distances between backbone atoms (N, CA, CB, C, O) and grouped them by atom types and residue number separation along the peptide chain. Separations above five residues were grouped together into one. For each of the 270 pairs of atom types and residue separation groups, we constructed a lookup table of with the average value, the minimum value (lower bound) and the maximum value (upper bound). Outliers, defined as distances outside the 1/1,000 percentile, were discarded in the upper and lower bound calculations, for robustness. Given a partial protein distance matrix, lower and upper bounds for unknown distances are set to the corresponding values in the lookup table.\n\nThe average, upper and lower bounds for C-alpha and C-beta distances are shown in Figure 2A. As expected, the uncertainty around the average distance increases with the residue separation. The lower bound, however, reaches a minimum value of 3.70Å for C-alpha and 3.53Å for C-beta contacts. Values below 3Å are observed for C-alpha atoms of adjacent residues (separation equal to 1), which correspond to cis-Proline conformations. In order to simplify the lookup table of atomic distance bounds, we discarded cis-Prolines. A subset of the final distance bounds lookup table for two adjacent residues is shown in Figure 2B. Dark colours in the table represent distances with low variation, for example those between atoms that form the peptide bond plane (CA0, C0, O0, N1, CA1).\n\nA) C-alpha (CA) and C-beta (CB) interatomic distances as a function of residue separation along the peptide chain. The average distances are shown as a black line with a grey area for lower and upper bounds. Upper bounds trimmed at 20Å . B) Table of average backbone interatomic distances between adjacent residues (0,1), excluding cis-Prolines. Colours indicate amount of variability as the difference between upper and lower bounds in Å, with darker colours for smaller variations.\n\nProteins with known pairs of native and rearranged structures have been selected from the Protein Data Bank (PDB) and used as modelling examples. The use of pairs has allowed the comparison of EDM models to their experimentally determined structures. For domain atrophy, a pair of close homologs were selected instead due to the lack of domain atrophied structures from the same protein.\n\nThe procedure to model structural rearrangements using EDMs is schematically shown in Figure 1 and starts by parsing the atomic coordinates of the domain structure from a PDB file and extracting a subset of atoms, either C-alpha or backbone atoms. Next, the distance matrix is calculated and its entries are rearranged, corresponding to the structural changes to be modelled. Unknown distances are bounded using the lookup table of atomic distance bounds and filled using EDM completion. Finally, the atomic coordinates of the rearranged structure are obtained using multidimensional scaling and substituted into the original PDB file, which is saved as the new model.\n\nThe EDM protein modelling approach presented here has been implemented in the R programming language, version 3.6. Protein structures are parsed and manipulated using the latest version (2.4) of the bio3d package15. The edmcr package version 1.016 is used for EDM completion and multidimensional scaling. The implementation requires minimal dependencies and only a few lines of code, and runs on a single CPU. A general script for each type of structural rearrangement has been created so that it can be applied to any query protein by simply changing the path to the input file.\n\n\nResults and Discussion\n\nA circular permutation of a protein domain is the alteration of its amino acid sequence order so that the N and C-terminal regions are interchanged at a specific \"cut\" position. The result is a new domain with a similar overall 3D structure and topology but different termini2.\n\nThe core of the domain remains largely the same, so atomic distances can be rearranged in the matrix, as shown in Figure 1. For a given native distance matrix D with n rows and columns, the distance matrix Dcp of a circular permutation at position k corresponds to:\n\nThe EDM completion algorithm will not converge to a solution if joining the domain termini is not geometrically possible. In that case, a longer linker or more unfolded terminal residues might have to be considered, as is the case for YibK methyltransferase. The distance between the N and C-terminal residues is 20Å, which means a linker is required to connect them. An experimental structure of a circular permuted YibK has five extra residues forming the termini linker loop17. Modelling the same circular permutation at position 82 with EDMs using only two unfolded terminal residues does not converge to a valid solution, with a large loss value over 20K and distance errors in the terminal loop region above 8Å. An EDM model with four unfolded residues neither converges to a solution, but has a smaller loss of 2K and errors on the order of 2Å. Using six unfolded terminal residues finally converges to an exact solution, shown in Figure 3A.\n\nA) C-alpha model of the circular permutation of YibK methiltransferase as a rainbow backbone, superposed to the native structure (PDB: 1MXI) as transparent cartoon; B) Domain swap dimer model of Cyanovirin-N (PDB: 2EZM) as a rainbow backbone, superposed to its experimental domain swap dimer (PDB: 3EZM) as transparent cartoon; C) Domain swap dimer model of EPS8 SH3 domain (PDB: 1I0C) as a rainbow backbone, superposed to the left-most domain of its experimental domain swap dimer (PDB: 1I07) as transparent cartoon; D) Backbone EDM model of domain atrophied Rib domain (PDB: 6SX1) from the structure of Rib Long (PDB: 6S5W); and E) C-beta distance matrices of monomeric EPS8 domain (PDB: 1I0C), its experimental domain swap dimer (PDB: 1I07) and its model generated with EDMs. Conserved intra-domain contacts in the monomer distance matrix are highlighted as red boxes, while contacts swapped to inter-domain are highlighted as orange boxes.\n\nDomain swapping refers to the reciprocal exchange of equivalent secondary structure elements between protein domains6. The swapping mechanism requires the extension of a short region of the native domain to form what is known as the \"hinge loop\" that bridges the two interacting domains. Although the core of the domain in a domain swap is formed by inter-domain interactions, as opposed to intra-domain interactions in the non-swapped conformation, its overall 3D structure is similar everywhere else apart from the \"hinge loop\" region. Therefore, like for circular permutations, atomic distances in the matrix can be rearranged to alter the pattern of residue interactions.\n\nFor a given distance matrix D from a monomeric domain with n atoms, the matrix Dsw for a domain swap with \"hinge loop\" centred at position k corresponds to a duplicated matrix with 2n columns and rows. Intuitively, interactions between atoms at each side of the \"hinge loop\" are changed to be inter-domain and interactions involving residues within each side are kept intra-domain, as shown in Figure 3E. This corresponds to the following matrix index rearrangements:\n\nThe structures of Cyanovirin-N and EPS8 SH3 domains have been experimentally determined both as monomeric and domain swapped dimers. The EDM model of the Cyanovirin-N domain swap structure from its monomer closely resembles the experimental domain swap at position 5118, as shown in Figure 3B. However, the EDM model of the EPS8 domain swap structure from its monomeric form converges to a valid solution, but has a different domain orientation to the experimental domain swap at position 3419, as shown in Figure 3C. These results imply that the domain swap of Cyanovirin-N has a lower flexibility than EPS8. The relative orientation between Cyanovirin-N domains is constrained and cannot change without disrupting natural protein geometry, while EPS8 domains have a greater range of possible orientations.\n\nTo model domain swap dimers, atomic coordinates can be directly assigned to two different chains. Another case is that of tandem domain swaps, where the two domains forming the swapped conformation are part of the same protein chain. In tandem domain swaps, an additional loop is required to connect the termini of the central domain at indices (n, n + 1), similar to the circular permutation case. The TAndem DOmain Swap Stability predictor (TADOSS) method9 already reports predictions for the \"hinge loop\" position and linker lengths required for the formation of tandem domain swaps. Hence, EDM modelling can be directly used to generate 3D models for TADOSS predictions. This feature has been implemented in the latest version of the method. The possibility to generate models for TADOSS predictions not only improves result visualisation and interpretability, but offers a new avenue to improve its accuracy.\n\nDomain swapping is also found as a crystallisation artefact20–22. By inverting the domain swap matrix rearrangement, it is possible to \"unswap\" these structures using EDMs, facilitating its interpretation and comparison to other known domain structures.\n\nDomain atrophy is the abrupt loss of entire secondary structures in the core of a protein domain, resulting in a shorter version of the domain3. Although it is a rare evolutionary event, it has been described for some of the most widespread and conserved domain folds, like immunoglobulins (Ig) and TIM barrels3,23.\n\nA structural deletion between positions k and l in a domain can be modelled using EDMs by removing all columns and rows between indices k and l in the native distance matrix. Since residues k and l are connected through a peptide bond in the atrophied domain, constraints for adjacent residues from the lookup table of distance bounds have to be applied to the distance matrix accordingly.\n\nOpposite to circular permutations and domain swaps, domain atrophy can have a more significant effect on the core of the domain, so it might have an impact on a higher fraction of native distances. As a result, the EDM completion is more likely to fail to converge to an exact solution. Proposed workarounds are to either \"unfold\" the residues around the deletion or to \"relax\" the native distances to intervals. Instead of using the exact distances, bounds can be set to a 10% interval of their native distances, for example. That way, the structure is given enough flexibility to adapt to the new conformation, while still conserving its overall 3D shape.\n\nThe domain atrophy in Rib domains involved the loss of a pair of beta strands core to its beta-sandwich topology23. The distance between the two ends of the deleted segment between positions 55 and 72 is, however, close enough so that a short linker can connect them, as captured by the EDM atrophy model shown in Figure 3D. The atrophy leaves, however, part of the domain core exposed to the surface. This gap is filled in the Rib domain by another part of the protein which adopts a helical turn conformation. This type of structural compensation is common in domain atrophy cases. Although such detailed structural consequences of domain atrophy can not be accounted for by EDM models, they provide a useful starting point to investigate them further.\n\nThe size of a distance matrix scales quadratically with the number of atoms in the input structure. This means that the running time and memory of EDM algorithms is expected to be at least 𝒪(n2).\n\nCalculation of distance matrices and multidimensional scaling operations are very fast, and take less than a second for the modelling examples presented in Figure 3. The matrix completion step is, however, the bottleneck, varying from just a few seconds to few minutes, heavily dependent on the size of the input matrix, but also on the number and complexity of unknown entries and a random convergence factor.\n\nRunning times for modelling structural rearrangements of several domain structures is shown in Figure 4. C-alpha models take less than a minute for up to 200 residues, which is within the size of an average protein domain and suitable for large-scale analysis. C-alpha models of domains up to 400 residues run in less than ten minutes. In comparison, backbone models do not scale particularly well, with an expected factor of 25 times slower than C-alpha models due to their five atoms per residue. Hence, backbone models for domains over 200 residues are possible but not practical for most applications.\n\nRunning times were calculated on a single standard CPU with 8GB of RAM. The small computational resources needed to create a model allows multiple modelling jobs to run concurrently in large-scale analyses.\n\nTime calculated for backbone and C-alpha models of circular permutations (CP) and domain swap dimers (DSD) of the 20 ECOD representative domains. Backbone models for domains over 300 residues are omitted. Quadratic fits of the form y = Ax2 for each model granularity are shown as lines with uncertainty grey shades. Running time shown as a square root scale.\n\n\nConclusions\n\nWe have described a new homology modelling approach for protein structures based on their distance matrices. The approach aims to be intuitive, flexible, light-weight and fast, being an alternative to more traditional modelling techniques based on simulation. Through a series of examples, we have demonstrated how EDMs can be handled to suit different modelling needs and its applications for protein structure analysis. We are also releasing an open code repository with an implementation of the method to the community, hoping that other researchers can explore the modelling cases presented in this study and extend the code for other applications.\n\nModelling protein structures with EDMs is well suited to tasks where a large proportion of atomic distances are known close to their exact values. As we have shown here, this is the case for structural rearrangements, where only a small subset of distances in the native matrix are perturbed. There are a number of other applications in protein modelling where this condition holds and that we have not explored here, including modelling of multidomain proteins by concatenating tandem or nested domains, docking protein subunits based on a subset of interface contacts, modelling allosteric conformational changes, and incorporating distance constraints from experimental techniques (NMR, cross-linking) and sequence co-evolution inferences.\n\nModelling protein structures using EDMs is far behind state-of-the-art techniques based on atomic energies and simulations, and therefore has its own limitations. EDM models are solely based on geometry, and it is not trivial how to incorporate other more complex energetic terms essential to protein structures. Additionally, there is a lack of specialised software libraries for EDM-based protein modelling, making it difficult to write efficient code for more sophisticated applications.\n\nThe current implementation in R does not scale well and proves impractical for inputs over 600 atoms, mainly due to the matrix completion bottleneck. This means most of the models are restricted to be coarse-grained (C-alpha or backbone only), even though the same approach described here can be used to generate full side-chain models. Adding residue side-chains to backbone models is, however, a common task in protein modelling and good tools already exist, for example Chimera’s Dock Prep24. The actual computational costs of EDM modelling can be reduced much further. On the software implementation side, matrix operations in R are two orders of magnitude slower than in other programming languages like C++ and Java. The use of an alternative library for faster EDM completion will have a big impact on the running time, but we are unaware of its existence. Algorithmically, the size of EDMs can be reduced exploiting rigid \"cliques\" of fully connected points. This technique is known as facial reduction and has been previously used to reduce the size of protein distance matrices12.\n\nDespite its current limitations, several protein modelling tasks can benefit from the EDM modelling approach described in this article. As shown for structural rearrangements, EDM models prove to be another valuable tool in the protein structure analysis toolbox.\n\n\nData availability\n\nZenodo: lafita/protein-edm-demo: Initial public release. https://doi.org/10.5281/zenodo.394538325 This project contains the following underlying data:\n\nprotein_bounds.tsv (atomic distance bounds in Figure 2)\n\nruntimes_ecod.tsv (running time data underlying Figure 4)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nProtein EDM modelling implementation in R.\n\nSource code available from: https://github.com/lafita/protein-edm-demo\n\nArchived source code at the time of publication: https://doi.org/10.5281/zenodo.394538325\n\nLicense: MIT license\n\nTADOSS method incorporating automatic generation of tandem domain swap models.\n\nSource code available from: https://github.com/lafita/tadoss\n\nArchived source code at the time of publication: https://doi.org/10.5281/zenodo.393915026\n\nLicense: MIT license", "appendix": "References\n\nRousseau F, Schymkowitz J, Itzhaki LS: Implications of 3D domain swapping for protein folding, misfolding and function. Adv Exp Med Biol. 2012; 747: 137–52. PubMed Abstract \| Publisher Full Text\n\nUliel S, Fliess A, Unger R: Naturally occurring circular permutations in proteins. Protein Eng. 2001; 14(8): 533–42. PubMed Abstract \| Publisher Full Text\n\nPrakash A, Bateman A: Domain atrophy creates rare cases of functional partial protein domains. Genome Biol. 2015; 16(1): 88. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIwakura M, Nakamura T, Yamane C, et al.: Systematic circular permutation of an entire protein reveals essential folding elements. Nat Struct Biol. 2000; 7(7): 580–585. PubMed Abstract \| Publisher Full Text\n\nYang S, Cho SS, Levy Y, et al.: Domain swapping is a consequence of minimal frustration. Proc Natl Acad Sci U S A. 2004; 101(38): 13786–13791. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLafita A, Tian P, Best RB, et al.: Tandem domain swapping: determinants of multidomain protein misfolding. Curr Opin Struct Biol. 2019; 58: 97–104. 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J Mol Biol. 2019; 431(4): 857–863. PubMed Abstract \| Publisher Full Text\n\nYang F, Bewley CA, Louis JM, et al.: Crystal structure of cyanovirin-N, a potent HIV-inactivating protein, shows unexpected domain swapping. J Mol Biol. 1999; 288(3): 403–412. PubMed Abstract \| Publisher Full Text\n\nKishan KV, Newcomer ME, Rhodes TH, et al.: Effect of pH and salt bridges on structural assembly: Molecular structures of the monomer and intertwined dimer of the Eps8 SH3 domain. Protein Sci. 2001; 10(5): 1046–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXiaozhen Hu, Wang H, Ke H, et al.: High-resolution design of a protein loop. Proc Natl Acad Sci U S A. 2007; 104(45): 17668–73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchlesier J, Rohde M, Gerhardt S, et al.: A Conformational Switch Triggers Nitrogenase Protection from Oxygen Damage by Shethna Protein II (FeSII). J Am Chem Soc. 2016; 138(1): 239–47. PubMed Abstract \| Publisher Full Text\n\nHara K, Taharazako K, Ikeda M, et al.: Dynamic feature of mitotic arrest deficient 2–like protein 2 (MAD2L2) and structural basis for its interaction with chromosome Alignment–maintaining phosphoprotein (CAMP). J Biol Chem. 2017; 292(43): 17658–17667. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWhelan F, Lafita A, Griffiths SC, et al.: Defining the remarkable structural malleability of a bacterial surface protein Rib domain implicated in infection. Proc Natl Acad Sci U S A. 2019; 116(52): 26540–26548. Publisher Full Text\n\nPettersen EF, Goddard TD, Huang CC, et al.: UCSF Chimera - A visualization system for exploratory research and analysis. J Comput Chem. 2004; 25(13): 1605–1612. PubMed Abstract \| Publisher Full Text\n\nLafita A: lafita/protein-edm-demo: Update license. 2020. http://www.doi.org/10.5281/zenodo.3945383\n\nLafita A: lafita/tadoss: Tadoss v1.1.2020. http://www.doi.org/10.5281/zenodo.3939150" }	[ { "id": "67901", "date": "20 Aug 2020", "name": "Sergey Ovchinnikov", "expertise": [ "Reviewer Expertise I work on developing methods for protein structure prediction." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFor this manuscript, the authors start by reviewing how Euclidean distance matrices (EDM) can be used to represent protein structures, highlighting the various pros of the approach. As an example, they highlight how EDM can be easily used to express and model circular permutations. Finally, they provide opensource R code.\nConcerns/Questions:\nOne reason why structural modelers avoid working with EDMs directly is due to Chirality. Traditionally, one would work with coordinates directly, and use EDMs to calculate pairwise decomposable energies. Though the authors mention that chirality can be easily resolved after reconstruction (assuming the entire structure is of the same chirality), they do not go into detail of how chirality is handled during matrix-completion. It seems without explicit term to promote the same chirality through the entire structure, matrix-completion followed by MDS could return a structure that alternates between different chiralities. Maybe due to the shortness of the loops modeled this is not an issue? One way to check would be to compute the backbone dihedrals. For example, we'd expect the most of the phi dihedral between atoms c-n-ca-c to be negative... if the new modeled loop is positive, this could indicate a problem.\n\nIn conclusion, the authors write: \"Modelling protein structures using EDMs is far behind state-of-the-art techniques based on atomic energies and simulations, and therefore has its own limitations.\" Given distances (aka EDMs) are used to represent pairwise decomposable terms in state-of-the-art techniques... it doesn't seem it would be that difficult to incorporate these energies in a technique that optimizes distance matrix values as opposed to coordinate values. Some energy terms that use distances include Lennard-Jones van der Waals, Coulomb electrostatics. Even angles and dihedrals could potentially be approximated as distances between i,i+2 and i,i+3. See Rosetta Energy function for reference: https://pubs.acs.org/doi/10.1021/acs.jctc.7b00125\n\nFor modeling circular permutations, it seems one could just modify cartesian coordinates directly. Circular permutations would be as easy moving xyz entries from beginning to end of the list. It's not clear what advantage EDM provides, besides enabling the use of some of the matrix-completion algorithms for determining the best atom placements. What are the specific advantages over simply minimizing/placing coordinates directly?\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? Yes", "responses": [] }, { "id": "67413", "date": "06 Nov 2020", "name": "Henry Wolkowicz", "expertise": [ "Reviewer Expertise Convex optimization", "numerical linear algebra." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverview This work focuses on how to formulate structural rearrangements of proteins as a Euclidean distance matrix completion (EDMC) problem with an homology modelling approach. The authors present three types of structural rearrangements that can be formulated as a EDMC problem; circular permutations, domain swapping and domain atrophy. These structural arrangements yield some missing entries in the Euclidean distance matrices. In order to fill in the missing entries, they adopt the dissimilarity parametrization formulation algorithm by Trosset. For the purpose of obtaining realistic conformations using EDM model, the authors engage bounds on missing values, where the bounds are obtained from some experimental data. They also suggest some modelling heuristics in order to accommodate some difficulties that give rise to algorithmic failure. The paper is well written with many interesting overviews of the underlying molecular problems, but with little of the mathematical background of EDM and of rigidity. The main concern is that the numerics use one approach from 2000, i.e., one approach that does not allow readers to know if it is competitive with other approaches of the time and more recent methods. And, how does the noise in the data influence the results?\nQuestions and Suggestions\nThere are many papers on using EDM for molecular conformation. First there is a recent book on EDM1 that emphasizes rigidity, a property that is important to exploit for molecular conformation and protein structure problems, as the backbone often is fixed and known, i.e., rigid.\n\nThe following is a quote from the submitted paper: ”The actual computational costs of EDM modelling can be reduced much further. On the software implementation side, matrix operations in R are two orders of magnitude slower than in other programming languages like C++ and Java”. If you know that the implementation using R is slower than other programming languages, what is the reason for choosing this particular language?\n\nThe following is a quote from the submitted paper: “Distance matrices can be inverted into a set of points in an embedding space of m dimensions using a technique known as multidimensional scaling (MDS)”. Some references would be nice so that readers who are not familiar with the subject can go find the relevant materials.\n\nThe only reason why the dissimilarity parametrization formulation (DPF) algorithm is chosen to solve the EDMC problem seems to engage the bounds on missing entries effectively. Are there any other reasons why the DPF is an appropriate choice of the algorithm? Are there any other algorithms or methods that appear in the literature that are able to handle this model? The approach by Trosset is from 20002. There are many other approaches and many more recent. For example for noisy problems, see reference 33 and the references therein, e.g., the papers by Alipanahi et al.,4.\n\nThe matrix rearrangement formula, appears in the middle of the second paragraph in domain swapping on page 4, is difficult to follow. A clearer and understandable description is required.\n\nIt is mentioned in Introduction that the structural rearrangements of interest have been extensively studied. However, in the numerical experiment presented in the submitted paper, no discussions on the comparative performances are made in order to address the strength of this new approach. Some discussions that engage the advantages of this approach over many other approaches are suggested.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes. It is clearly explained but there is no proper rational for using only ONE method without any comparison with other methods. There are many other techniques around for (noisy) EDM completion problems\n\nIs the description of the method technically sound? Yes. The paper is well written and results/method well explained.\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. The references for the data that is used is provided.\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? As mentioned above only one method (from 2000) is used. Moreover, the details of the influence of noise in the data is NOT clearly explained.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-728
https://f1000research.com/articles/8-1937/v1	20 Nov 19	{ "type": "Research Article", "title": "SCIMITAR+ Trial: A randomised study within a trial (SWAT) of a contingent financial reward to improve trial follow-up", "authors": [ "Catherine Arundel", "Elizabeth Coleman", "Caroline Fairhurst", "Emily Peckham", "Della Bailey", "Simon Gilbody", "Elizabeth Coleman", "Caroline Fairhurst", "Emily Peckham", "Della Bailey", "Simon Gilbody" ], "abstract": "Background: To evaluate the effectiveness of a contingent financial incentive (£10 note in addition to a routinely provided £10 voucher) versus no contingent financial incentive, on improving the retention rate in a randomised controlled trial (RCT). Methods: A two arm ‘Study within a Trial’ (SWAT) embedded within a host RCT (SCIMITAR+). Participants were randomised to the SWAT using a 2:1 (intervention:control) allocation ratio. The primary outcome measure was the proportion of participants completing a CO breath measurement at the first SCIMITAR+ follow up time point (6 months). Secondary outcomes were withdrawing from follow-up after contact and time from assessment due date to completion. Analyses were conducted using logistic or Cox Proportional Hazards regression as appropriate. Results: A total of 434 participants were randomised into this SWAT. Completion of the CO breath measurement at 6 months was 88.5% (n=247) in the intervention arm of the SWAT and 85.4% (n=123) in the control arm. The difference (3.1%) was not statistically significant (p=0.36; OR 1.29, 95% CI 0.71-2.33, p=0.41). There was also no evidence of a difference in the proportion of participants withdrawing from follow-up after contact (intervention n=7 (2.5%), control n=5 (3.5%); OR 0.76, 95% CI 0.23-2.44, p=0.64), nor in terms of proximity of 6-month visit completion to due date (HR 1.07, 95% CI 0.86-1.33, p=0.55). Conclusion: Contingent financial incentives did not statistically significantly increase rates of face-to-face follow-up completion within the SCIMITAR+ trial population. However, the sample size of this SWAT was constrained by the size of the host trial and power was limited. This SWAT adds to the body of evidence for initiatives to increase response rates in trials.", "keywords": [ "SWAT", "retention", "randomized controlled trial" ], "content": "Introduction\n\nAttrition is a major problem for randomised controlled trials (RCTs) with 25% experiencing more than 10% attrition1.\n\nBower et al. (2014)2 identified financial incentives as an effective retention strategy (RR 1.18; 95% CI 1.09 to 1.28), and effectiveness was increased if this incentive was provided on receipt of a completed questionnaire (RR 1.25; 95% CI 1.14 to 1.38). Bailey et al. (2013)3 identified that varying the incentive level (£20 compared to £10) increased response to postal questionnaires by up to 10%.\n\nThis SWAT evaluated the effectiveness of a contingent financial reward - £10 cash in addition to a routinely provided £10 voucher - versus no contingent financial reward, on improving the retention rate in the SCIMITAR+ trial.\n\n\nMethods\n\nThis SWAT was embedded within the SCIMITAR+ RCT which evaluated the effectiveness of a bespoke, individually-tailored, smoking cessation programme, compared to usual care, for adult smokers with severe mental ill health conditions4. The SCIMITAR+ Trial was registered prospectively: ISRCTN72955454\n\nThis paper refers to the methods and results of the SWAT only.\n\nThe SWAT5 was conducted in 21 NHS Trusts and 16 primary care settings and was implemented after the start of SCIMITAR+ follow-up. Participants were eligible for this SWAT if they reached the SCIMITAR+ 6-month follow-up on or after 31st September 2016.\n\nWhen participants in the SWAT intervention group were contacted by the research team to arrange their follow-up appointment, they were advised of the potential of receiving £10 cash contingent on providing a carbon monoxide (CO) breath measure as part of their 6-month face-to-face study appointment, in addition to the £10 gift voucher routinely provided to all participants. Participants in both groups received all other pre-planned retention strategies within SCIMITAR+.\n\nThe primary outcome for the SWAT was the proportion of participants completing a CO breath measurement at the SCIMITAR+ 6 month follow-up time-point. Secondary outcome measures were: i) the proximity of visit completion to visit due date; ii) the proportion of participants withdrawing from follow-up in the two months after initial contact was made to arrange the 6-month visit.\n\nThe sample size was determined by the number of participants followed-up at 6 months in SCIMITAR+ from the point at which this SWAT was embedded.\n\nSimple randomisation using random numbers was carried out by an independent statistician at the York Trials Unit using Stata v136. Participants were allocated with a 2:1 allocation ratio (intervention:control) due to the anticipated effectiveness of financial incentives increasing questionnaire response rates.\n\nIt was not possible to blind research staff to the participant’s allocation. Participants were not informed about the SWAT so were blind to the study hypothesis.\n\nThe SWAT was approved by the Research Ethics Committee Yorkshire and Humber – Leeds East (15/YH/0051). As the SWAT was deemed to be low risk, and to avoid disappointment for participants who did not receive the additional incentive, informed consent was not obtained for participation in this SWAT.\n\nAnalyses were conducted using Stata v157 on an intention to treat basis using two-sided statistical tests at the 5% significance level, adjusting for host trial allocation.\n\nThe proportion of participants who provided a 6-month CO breath measure was analysed using logistic regression. The odds ratio (OR), 95% confidence interval (CI) and p-value are presented.\n\nThe 6-month appointment due date was 183 days after randomisation. Participants who withdrew a month either side of the 6-month appointment due date were classed as withdrawn. The proportion of participants withdrawing from SCIMITAR+ in the two months after contact were analysed in the same way as the primary outcome.\n\nA Cox Proportional Hazard model compared the proximity of the visit completion to visit due date (time in days). Participants who completed their visit before or on the due date had their time-to-visit set to 0.1.\n\n\nResults\n\nIn total, 434 participants were randomised into this SWAT (n=286, 65.9% intervention group; n=148, 34.1% control group). Eleven participants withdrew from SCIMITAR+ following randomisation but prior to being contacted for their 6-month visit and were excluded from analysis. There were 423 eligible participants (intervention group n=279, 66.0%; control group n=144, 34.0%) (Figure 1).\n\nOverall, 87.5% (n=370) of participants completed the CO breath measurement at 6 months; there was no statistically significant difference between intervention (88.5%, n=247) and control groups (85.4%, n=123) (3.1% difference, OR 1.29, 95% CI 0.71-2.33, p=0.41). There was no significant difference in withdrawals between trials arms (intervention n=7, 2.8%; control n=5, 3.5%; OR 0.76, 95% CI 0.23-2.44, p=0.64) or proximity of 6-month visit completion to due date (hazard ratio 1.07, 95% CI 0.86-1.33, p=0.55).\n\n\nDiscussion\n\nAn additional £10 in cash did not statistically significantly increase the likelihood of participants completing a face-to-face follow-up, the proportion of the participants withdrawing, or have an effect on the proximity of the visit to the due date.\n\nA small positive difference was observed; however, despite the large sample size, the study was underpowered to confidently rule out a small ‘true’ effect. Due to the small effect size (3.1% increase in response) the cost per additional person attending would be in excess of £300.\n\nDue to the sample size of this SWAT, it is most likely generalisable to the larger host trial population of patients with severe mental ill health disorders.\n\nData was not collected on how study staff followed the guidance on discussing the contingent £10 note to intervention group participants when arranging follow up visits. This may have diluted the effect of the intervention.\n\n\nConclusion\n\nContingent financial incentives did not statistically significantly increase rates of face-to-face follow-up completion in this trial. However, there were sample size and power limitations. Future SWATs are needed to add to the evidence base.\n\n\nData availability\n\nFigshare: SCIMITAR+ Trial: A randomised study within a trial (SWAT) of a contingent financial reward to improve trial follow-up - Data Set, https://doi.org/10.6084/m9.figshare.10060202.v28.\n\nFigshare: CONSORT checklist for SCIMITAR+ Trial: A randomised study within a trial (SWAT) of a contingent financial reward to improve trial follow-up, https://doi.org/10.6084/m9.figshare.10060202.v28.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe SCIMITAR+ team acknowledge the support of the PROMETHEUS programme (MRC MR/R013748/1) in developing this manuscript.\n\n\nReferences\n\nHewitt CE, Kumaravel B, Dumville JC, et al.: Assessing the impact of attrition in randomized controlled trials. J Clin Epidemiol. 2010; 63(11): 1264–70. PubMed Abstract \| Publisher Full Text\n\nBower P, Brueton V, Gamble C, et al.: Interventions to improve recruitment and retention in clinical trials: a survey and workshop to assess current practice and future priorities. Trials. 2014; 15: 399. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBailey JV, Pavlou M, Copas A, et al.: The Sexunzipped trial: optimizing the design of online randomized controlled trials. J Med Internet Res. 2013; 15(12): e278. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeckham E, Arundel C, Bailey D, et al.: Smoking Cessation Intervention for Severe Mental Ill Health Trial (SCIMITAR+): study protocol for a randomised controlled trial. Trials. 2017; 18(1): 44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSWAT 48 – Effects of a £ 10 note on retention. Reference Source\n\nStataCorp: Stata Statistical Software: Release 13. College Station, TX: StataCorp LP. 2013.\n\nStataCorp: Stata Statistical Software: Release 15. College Station, TX: StataCorp LLC. 2017.\n\nArundel C, Coleman E, Fairhurst C, et al.: SCIMITAR+ Trial: A randomised study within a trial (SWAT) of a contingent financial reward to improve trial follow-up - Data Set. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.10060202.v2" }	[ { "id": "56823", "date": "03 Dec 2019", "name": "Frances Shiely", "expertise": [ "Reviewer Expertise Epidemiology", "trial methodological reserach" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this SWAT. I am in favour of publishing negative results as well as positive results, so my reason for rejecting this SWAT is not because the findings are negative. I wish to be clear on that.\n\nThis was a SWAT within the SCIMITAR+ trial. The purpose of the SWAT was to establish if £10 cash + £10 voucher would increase retention rates when compared to £10 voucher only. SCIMITAR+ was a smoking cessation trial. The authors conclude that contingent financial incentives did not statistically significantly increase rates of face-to-face follow-up completion within the SCIMITAR+ trial.\nIn the background section of the abstract, you describe the SWAT as “…versus no contingent financial incentive” which is misleading. There is a contingent financial incentive because you have a £10 voucher as standard. I presume it’s contingent on them turning up to the appointment? I had to go to the body of the paper to ensure I had the correct interpretation. You should describe the SWAT more accurately as “to establish if £10 cash + £10 voucher would increase retention rates when compared to £10 voucher only” and change this in the main body of the paper also.\nI struggled with the design of this SWAT and what the investigators hoped to achieve by it. I don’t think the primary outcome was suitable to measure what they intended. The conclusion drawn in the abstract is unrelated to the primary outcome. The purpose of the SWAT was to look at retention. The participants were eligible for the SWAT if they reached the SCIMITAR+ 6-month follow-up. I’d like the authors to comment on this in relation to their SWAT. It could be argued that if they were already coming for the 6 month follow-up they were motivated to continue regardless of the extra financial incentive. It seems like 100% of those contacted for the 6-month follow-up turned up to their appointment. Therefore, 100% were retained. I don’t see the point then in making the primary outcome contingent on the CO breath measurement and was not surprised there was no statistically significant difference between the two groups. Your secondary outcome, the proportion withdrawing after the contact would be more relevant. This is supported by the fact you used the proportion turning up for the appointment as your concluding statement.\nComparing two incentivised arms at 6 month follow-up doesn’t appear to be the best use of the money. What would have been interesting to find out is if the additional money at 12 months would make a difference to retention? Perhaps let the intervention arm know at 6-months that at 12 months, in addition to the voucher they would get £10 cash. Then you could establish if the additional incentive was effective.\n\nI would have liked to know what you told the comparator group when you phoned them to come to their 6-month visit. Did you tell them they were getting a £10 voucher to give a CO. Did you make them aware of what the intervention group were getting or did you tell them about the SWAT at all?\nThe participants were already incentivised with the £10 voucher. What is the evidence base for thinking that an additional £10 cash would increase retention at 6 months? I note the Bailey et al. 2013 study but this was in relation to response rates for a postal questionnaire.1\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5622", "date": "17 Jul 2020", "name": "Catherine Arundel", "role": "Author Response", "response": "In the background section of the abstract, you describe the SWAT as “…versus no contingent financial incentive” which is misleading. There is a contingent financial incentive because you have a £10 voucher as standard. I presume it’s contingent on them turning up to the appointment? I had to go to the body of the paper to ensure I had the correct interpretation. You should describe the SWAT more accurately as “to establish if £10 cash + £10 voucher would increase retention rates when compared to £10 voucher only” and change this in the main body of the paper also. We thank the reviewer for this helpful suggestion. We have amended the manuscript to make clearer the control group. I struggled with the design of this SWAT and what the investigators hoped to achieve by it. I don’t think the primary outcome was suitable to measure what they intended. The conclusion drawn in the abstract is unrelated to the primary outcome. The purpose of the SWAT was to look at retention. The participants were eligible for the SWAT if they reached the SCIMITAR+ 6-month follow-up. I’d like the authors to comment on this in relation to their SWAT. It could be argued that if they were already coming for the 6 month follow-up they were motivated to continue regardless of the extra financial incentive. It seems like 100% of those contacted for the 6-month follow-up turned up to their appointment. Therefore, 100% were retained. I don’t see the point then in making the primary outcome contingent on the CO breath measurement and was not surprised there was no statistically significant difference between the two groups. Your secondary outcome, the proportion withdrawing after the contact would be more relevant. This is supported by the fact you used the proportion turning up for the appointment as your concluding statement. We thank the reviewer for their comments here and apologise for any confusion. Having reviewed, we believe that the conclusion does relate to the primary outcome given it was unclear, due to the wide confidence intervals if there was any difference in completion of a CO breath measure between the two groups. Following review of this, and comments from Reviewer 2, we can see there has been some misunderstanding. The study randomisation was done in a single batch, rather than when the participant reached the Month 6 time point, therefore at the time of randomisation it was no known if the participant would attend their 6 month visit. We have made this clearer in the randomisation and results sections. We are clear how the reviewer has derived this information. Figure 1 demonstrates that 32 intervention and 21 control participants did not attend a face to face visit and so did not provide a CO breath measure. Comparing two incentivised arms at 6 month follow-up doesn’t appear to be the best use of the money. What would have been interesting to find out is if the additional money at 12 months would make a difference to retention? Perhaps let the intervention arm know at 6-months that at 12 months, in addition to the voucher they would get £10 cash. Then you could establish if the additional incentive was effective. The impact of the contingent reward on subsequent follow up time points is an important consideration. The effects were not assessed in this study, but this would be important to include in any subsequent studies of this nature. I would have liked to know what you told the comparator group when you phoned them to come to their 6-month visit. Did you tell them they were getting a £10 voucher to give a CO. Did you make them aware of what the intervention group were getting or did you tell them about the SWAT at all? As noted in the intervention description, during the call to arrange a follow up appointment intervention participants were advised of the contingent £10 if they were willing to meet face to face and provide the primary outcome. The intervention section has been reviewed to make this clearer. The participants were already incentivised with the £10 voucher. What is the evidence base for thinking that an additional £10 cash would increase retention at 6 months? I note the Bailey et al. 2013 study but this was in relation to response rates for a postal questionnaire.1 There is much available evidence on strategies to improve postal questionnaire response rates, but limited evidence for strategies to improve face to face visit completion. We suggest that strategies used for postal questionnaire response rates may have similar effectiveness for face to face visits, hence the completion of this SWAT. We have made clear in the background the justification for this." } ] }, { "id": "62472", "date": "24 Apr 2020", "name": "Sarah Rhodes", "expertise": [ "Reviewer Expertise Statistics", "randomised trials", "embedded trials", "meta-analysis." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThere seems to be an error in this sentence in the abstract – ‘The difference (3.1%) was not statistically significant (p=0.36; OR 1.29, 95% CI 0.71-2.33, p=0.41).’ – two different p-values are reported and the incorrect one should be removed.\n\nThis is a paper about the financial incentive SWAT and not the SCIMITAR+ trial – the title seems to suggest that this is the trial report for the SCIMITAR+ trial. Something like ‘The effect of contingent financial reward to improve retention; A randomised study embedded within the SCIMITAR+ trial’ might be better.\n\nThis SWAT has minimal power to answer a research question by itself, but as the authors rightly say it is an important addition to the body of evidence. I find the term ‘not statistically significant’ potentially misleading because it seems to suggest that the intervention wasn’t effective whereas the confidence intervals are very wide, and appear consistent with previous results. I’d recommend removing reference to ‘statistical significance’ and instead using terms like ‘it is unclear whether….’, given the very wide confidence intervals.\n\nThe description ‘This SWAT evaluated the effectiveness of a contingent financial reward - £10 cash in addition to a routinely provided £10 voucher - versus no contingent financial reward, on improving the retention rate in the SCIMITAR+ trial’ is difficult to understand. It seems like the intervention is the offer of a contingent financial reward rather than the reward itself (which happens after outcome measurement) – should this perhaps be ‘the effectiveness of the offer of a contingent financial reward’? The £10 voucher happens in both arms so perhaps this should say ‘versus the routinely rewarded £10 voucher alone’ – unless I’m misunderstanding what the control group is here.\n\nIt says ‘contingent financial reward’ in some places and ‘financial incentive’ in others – are these the same thing? It will be helpful if one term were used consistently.\n\nIt says that analyses were ‘on an intention to treat basis’ but 11 randomised participants that withdrew before the 6-month visit were excluded from the analysis; this is not really compatible with a true ‘intention to treat’ analysis which aims to preserve randomised groups. Perhaps clearly describe the analysis population rather than calling it ‘intention to treat’ – e.g. the analysis population was participants randomised to the SWAT who had not withdrawn by time X days before due visit. Consider a sensitivity analysis including all randomised participants.\n\nThe observed effect size was 3.1%; there is lots of uncertainty in this estimate so ideally it shouldn’t really be used to infer the likely cost effectiveness of future studies – instead, I’d recommend considering each end of the confidence interval (or perhaps the upper end as a best-case scenario) when considering cost per additional person retained.\n\nI don’t understand the sentence ‘Due to the sample size of this SWAT, it is most likely generalisable to the larger host trial population of patients with severe mental ill health disorders.’ A large sample size does not guarantee generalisability, and as noted the sample size is not really large enough to draw conclusions about this population. I think this point needs to be constructed more clearly.\n\nFor the secondary outcome of ‘proximity to due date’, I’m really interested to know whether the proportional hazards assumption holds. It would be helpful to include a Kaplain-Meier curve and/or some comment about assumption checking.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5623", "date": "17 Jul 2020", "name": "Catherine Arundel", "role": "Author Response", "response": "There seems to be an error in this sentence in the abstract – ‘The difference (3.1%) was not statistically significant (p=0.36; OR 1.29, 95% CI 0.71-2.33, p=0.41).’ – two different p-values are reported and the incorrect one should be removed.We thank the reviewer for this observation. The error has been corrected accordinglyThis is a paper about the financial incentive SWAT and not the SCIMITAR+ trial – the title seems to suggest that this is the trial report for the SCIMITAR+ trial. Something like ‘The effect of contingent financial reward to improve retention; A randomised study embedded within the SCIMITAR+ trial’ might be better.We thank the reviewer for this observation. The title has been amended for clarity.This SWAT has minimal power to answer a research question by itself, but as the authors rightly say it is an important addition to the body of evidence. I find the term ‘not statistically significant’ potentially misleading because it seems to suggest that the intervention wasn’t effective whereas the confidence intervals are very wide, and appear consistent with previous results. I’d recommend removing reference to ‘statistical significance’ and instead using terms like ‘it is unclear whether….’, given the very wide confidence intervals.We agree with the reviewers thoughts here. The manuscript has therefore been updated to remove ‘statistical significance’ and to make clear the lack of certainty. P values have however been retained for completeness.The description ‘This SWAT evaluated the effectiveness of a contingent financial reward - £10 cash in addition to a routinely provided £10 voucher - versus no contingent financial reward, on improving the retention rate in the SCIMITAR+ trial’ is difficult to understand. It seems like the intervention is the offer of a contingent financial reward rather than the reward itself (which happens after outcome measurement) – should this perhaps be ‘the effectiveness of the offer of a contingent financial reward’? The £10 voucher happens in both arms so perhaps this should say ‘versus the routinely rewarded £10 voucher alone’ – unless I’m misunderstanding what the control group is here.We have amended the manuscript in line with the reviewer’s suggestion to make clearer the control group.It says ‘contingent financial reward’ in some places and ‘financial incentive’ in others – are these the same thing? It will be helpful if one term were used consistently.We apologise for this inconsistency. The manuscript has been updated throughout to ensure consistency,It says that analyses were ‘on an intention to treat basis’ but 11 randomised participants that withdrew before the 6-month visit were excluded from the analysis; this is not really compatible with a true ‘intention to treat’ analysis which aims to preserve randomised groups. Perhaps clearly describe the analysis population rather than calling it ‘intention to treat’ – e.g. the analysis population was participants randomised to the SWAT who had not withdrawn by time X days before due visit. Consider a sensitivity analysis including all randomised participants.We can see why the reviewer feels that this is not strictly ITT. The study randomisation was done in a single batch, rather than when the participant reached the Month 6 time point, and so participants received an allocation when they were not technically eligible for the SWAT, hence the exclusion. As a result, we suggest that the analysis in the paper is ITT in the sense that the analysis includes everyone who would have been eligible for the SWAT. We have sought to make this clearer in the paper for avoidance of confusion. For added confidence, the analysis has been re-run including those who withdrew post randomisation, but pre visit. When these are included the findings do not change. The observed effect size was 3.1%; there is lots of uncertainty in this estimate so ideally it shouldn’t really be used to infer the likely cost effectiveness of future studies – instead, I’d recommend considering each end of the confidence interval (or perhaps the upper end as a best-case scenario) when considering cost per additional person retained.We thank the reviewer for making this important point.As the manuscript states, the cost of retaining an additional participant would be over £300. This statement is still correct in the worst case scenario, and as such covers the uncertainty; whilst also covering the estimate that we have found in this study. I don’t understand the sentence ‘Due to the sample size of this SWAT, it is most likely generalisable to the larger host trial population of patients with severe mental ill health disorders.’ A large sample size does not guarantee generalisability, and as noted the sample size is not really large enough to draw conclusions about this population. I think this point needs to be constructed more clearly.For clarity, this section has been amended to reflect that results are only applicable to the study population.For the secondary outcome of ‘proximity to due date’, I’m really interested to know whether the proportional hazards assumption holds. It would be helpful to include a Kaplain-Meier curve and/or some comment about assumption checking.The results section has been updated to detail assessment of hazards assumptions." } ] } ]	1	https://f1000research.com/articles/8-1937
https://f1000research.com/articles/9-618/v1	16 Jun 20	{ "type": "Research Article", "title": "Automatic migraine classification using artificial neural networks", "authors": [ "Paola A. Sanchez-Sanchez", "José Rafael García-González", "Juan Manuel Rúa Ascar", "José Rafael García-González", "Juan Manuel Rúa Ascar" ], "abstract": "Background: Previous studies of migraine classification have focused on the analysis of brain waves, leading to the development of complex tests that are not accessible to the majority of the population. In the early stages of this pathology, patients tend to go to the emergency services or outpatient department, where timely identification largely depends on the expertise of the physician and continuous monitoring of the patient. However, owing to the lack of time to make a proper diagnosis or the inexperience of the physician, migraines are often misdiagnosed either because they are wrongly classified or because the disease severity is underestimated or disparaged. Both cases can lead to inappropriate, unnecessary, or imprecise therapies, which can result in damage to patients’ health. Methods: This study focuses on designing and testing an early classification system capable of distinguishing between seven types of migraines based on the patient’s symptoms. The methodology proposed comprises four steps: data collection based on symptoms and diagnosis by the treating physician, selection of the most relevant variables, use of artificial neural network models for automatic classification, and selection of the best model based on the accuracy and precision of the diagnosis. Results: The neural network models used provide an excellent classification performance, with accuracy and precision levels >97% and which exceed the classifications made using other model, such as logistic regression, support vector machines, nearest neighbor, and decision trees. Conclusions: The implementation of migraine classification through neural networks is a powerful tool that reduces the time to obtain accurate, reliable, and timely clinical diagnoses.", "keywords": [ "artificial neural networks", "migraine", "supervised learning", "automatic classification techniques" ], "content": "Introduction\n\nCephalalgia or headache represents one of the most common types of pain experienced by humans. Headaches usually occur intermittently. The most frequent forms correspond to migraine and tension headache. Migraines are classified as chronic disorders of the nervous system and are characterized by the onset of recurrent symptoms or episodes associated with headache, which can range from moderate to severe pain and includes throbbing or vibrating pain; furthermore, migraines can be experienced unilaterally or bilaterally and can trigger other symptoms, such as nausea, vomiting, weakness, and light and sound sensitivity (Charles, 2013; Deza, 2010; IHS, 2018).\n\nBoth chronic and recurrent/relapsing headaches can cause pain and distress, but they rarely reflect a serious health problem. However, any change in the pattern or nature of the headache could be a sign of a serious complication, i.e., change in pain frequency from sporadic to frequent or pain severity from mild to acute; hence, medical attention should be sought as soon as possible (Goadsby et al., 2002).\n\nAlthough headache is generally a benign and transitory disorder that in most cases ceases spontaneously or with the aid of analgesics, it can also be caused by a serious life-threatening illness such as meningitis, brain tumor, hypercholesterolemia, heart problems, or subarachnoid hemorrhage (arteriovenous malformation). On the other hand, certain types of headaches, such as migraines, although benign, cause much suffering in affected individuals and represent an economic burden because of the high number of work-loss hours they cause (Trillos, 2010).\n\nIn 1988, the Classification Committee of the International Headache Society (IHS, 2018) published the current classification of headache types, which divides headaches into primary and secondary headaches. Primary headaches include migraines, tension-type headaches, paroxysmal headaches (cluster headaches and paroxysmal hemicrania), and benign miscellaneous headaches. Secondary headaches are those caused by vascular disease, infection, tumors, alteration in cerebrospinal fluid production, cranial trauma, neuralgia, etc.\n\nDiscrimination among migraines with and without aura and other types of migraines and headaches is established based on the specific criteria established by the International Headache Society (Charles, 2018; IHS, 2018; Rasmussen & Olesen, 1992; Viana et al., 2017) as follows:\n\nA. At least five attacks that meet criteria B to D.\n\nB. Duration from 4 to 72 hours.\n\nC. At least two of the following symptoms: 1. unilateral pain, 2. throbbing pain, 3. moderate-to-severe pain, 4. pain increases with physical activity.\n\nD. During headache, at least one of the following symptoms: 1. nausea and/or vomiting, 2. photophobia or phonophobia.\n\nE. At least one of the following: 1. Medical history that does not suggest secondary headache; 2. clinical history that suggests structural injury but is discarded via appropriate investigation; 3. structural injury exists, but the headache is not related with its presence nor is it related to time.\n\nA. At least two attacks that meet criterion B.\n\nB. At least three of the following four characteristics should be met: 1. One or more aura symptoms indicating focal cortical injury and/or brainstem dysfunction; 2. At least one gradually developing aura symptom lasting longer than 4 minutes or two or more successive symptoms; 3. The aura should not last for more than 60 minutes. If there is more than one aura, the duration of its presentation is proportional; 4. Headaches follow aura at intervals no greater than 60 minutes. Pain can be experienced before the aura or at the time of the aura.\n\nC. At least one of the following characteristics should be met: 1. Clinical history and physical and neurological examination that does not suggest secondary structural injury or metabolic disease; 2. If the clinical history or physical or neurological examination suggest secondary injury, this should be discarded via appropriate investigation; 3. In the presence of secondary injury, this does not explain the pain, it has no temporal relationship, and it does not occur for the first time.\n\nCauses: Many researchers agree that migraines have a genetic cause; however, there are a number of triggering factors, including stress; anxiety; hormonal imbalances in women; exposure to bright or flashing lights, loud noises, and strong odors; medications; inappropriate amount of sleep; sudden weather or environmental changes; overexertion (too much physical activity); tobacco or caffeine intake (consumption or withdrawal); skipping meals; medication overuse (taking migraine medication too often); and certain foods and food additives, such as alcohol, chocolate, ripened cheese, monosodium glutamate, some fruits and nuts, fermented or pickled products, yeast, and cured or processed meats (Evans, 2009).\n\nEpidemiological studies examining various types of populations show that people suffering from migraines share a very hectic social and work life, and as these individuals do not usually observe orderly resting times or practice other activities that help clear the mind, they are under stress, with migraine being a sign of work and emotional burden (Dodick, 2018; Parikh & Silberstein, 2019). Migraines also cause indirect damage to companies because they involve loss in labor productivity; annual losses in the United States are estimated at billions of dollars; such effects also apply to countries such as Colombia (Burch et al., 2018; Deza, 2010; Ramírez & Urrea, 2012).\n\nPhases: Migraines generally involve four phases (Burch, 2019; Katsarava et al., 2012):\n\n▪ Prodromic phase (previous): This phase begins up to 24 hours before you have a migraine. Early signs and symptoms include food cravings, unexplained mood swings, uncontrollable yawning, fluid retention, and increased urination.\n\n▪ Aura: If you are in this phase, you may see flashing or bright lights or criss-crossing lines. You may experience muscle weakness or a feeling of being touched or grabbed. Aura can occur just before or during a migraine.\n\n▪ Headache phase: In general, a migraine gradually begins and then becomes more severe. It often causes throbbing or vibrating pain, usually unilaterally. However, you can experience a migraine without experiencing a headache. Other migraine symptoms may include increased sensitivity to light, noise, and odor; nausea and vomiting; pain that worsens with movement; coughing; and sneezing.\n\n▪ Postdromal phase (after headache): You may feel exhausted, weak, and confused after a migraine. This can last up to 1 day.\n\nMigraines are more common during mornings. People often wake up suffering from migraines. Others experience migraines at predictable times, such as before menstruation or on weekends after a stressful work week (Kelman, 2007).\n\nDiagnosis: To diagnose migraines, the patient’s medical history and symptoms are assessed and a physical and neurological examination is performed; these are sometimes accompanied by specialized examinations such as magnetic resonance imaging, tomography, electroencephalogram, and lumbar puncture (Evans, 2019). An important part of diagnosing migraines is discarding other medical conditions that could be causing the symptoms (Altintop et al., 2017; Diamond et al., 2007; Giffin et al., 2003; Goadsby & Holland, 2019; Karsan & Goadsby, 2018; Maniyar et al., 2015).\n\nTreatment: There is no cure for migraines; therefore, treatment focuses on relieving symptoms and preventing further attacks. There are various types of medicines to relieve symptoms, such as triptans, ergotamine, and painkillers. The sooner these medications are administered, the more effective they are. In addition, to relieve symptoms, you can rest with your eyes closed in a quiet and dark room, place a cold cloth or ice pack on your forehead, or drink liquids (Burch, 2019; Charles, 2018; Diamond et al., 2007; Evans, 2009; Viana et al., 2017). Similarly, lifestyle changes can be adopted to control stress or other triggering factors. Furthermore, natural treatments can be used (May & Schulte, 2016).\n\nSome studies report that approximately 15% of United States citizens and 12% of the world’s population suffer from migraines (Burch, 2019; Burch et al., 2018; Chen et al., 2019; Diener et al., 2012; Dodick, 2018; Parikh & Silberstein, 2019). Migraines can affect anyone, but their prevalence increases in women, i.e., women are three times more likely to suffer from migraines than men. Migraines also have a high prevalence in those with a family history of migraines or those who suffer from medical conditions such as depression, anxiety, bipolar disorder, sleep problems, and epilepsy.\n\nIsaza et al. (1997) conducted a statistical study in mid-1981 and 1989 in Colombia and showed that 11.6% of women and 3.4% of men suffered from migraines. Similarly, Ramírez & Urrea (2012) reported that in 1997, of 3,401 patients assessed in Colombia in the outpatient department of the neurology service, 848 (24.93%) were due to primary headache, which is an important reason for outpatient consultation. Migraines occurred in 617 (18.14%) patients, with aura in 255 (7.5%) and no aura in 362 (10.64%).\n\nPrevious studies on migraine classification focused on the neurological or genetic aspects of the disease, leading to investigations that allowed for the classification of various types of migraines based on the study of encephalograms (Akben et al., 2012; Akben et al., 2010; Akben et al., 2016; Alkan & Akben, 2011; Altintop et al., 2017; Bellottia et al., 2007; Martins-Oliveira et al., 2017; Subasi et al., 2018), signals emitted by body temperature sensors, blood oxygen, heart rate, and electrodermal activity data (Chong et al., 2017; Koskimäki et al., 2017; Schwedt, 2013), or genetic analysis (Gormley et al., 2016). Although these studies attempted to classify migraines with precision levels of >70%, the methods used required a direct measurement of the variables using medical devices connected to the patient, which can cause variation in the data; lead to long waiting times for the appointment of specialized examinations, particularly in Latin American countries; or result in a lack of availability of medical equipment in rural regions. As stated previously (Alkan & Akben, 2011), numerous investigations have attempted to develop automatic diagnostic methods for migraines. However, no definitive diagnostic method for migraines has yet been accepted by the authorities on the subject (IHS).\n\nCurrently, there is a recurrent problem in the diagnosis and treatment of migraines, which includes, among others, the following needs: (i) proper reading and identification of the patient’s primary and secondary symptoms, (ii) precise and timely identification of the type of migraine, (iii) continuous monitoring of the symptoms, and (iv) adequate treatment. In the early stages of the pathology, patients visit the emergency services or outpatient consultation departments, where timely identification largely depends on the expertise of the treating physician and the continuous monitoring of the patient (Burch et al., 2018; Burch, 2019; Deza, 2010; Evans & Johnston, 2011; Trillos, 2010; Wang et al., 2019). However, owing to the scarcity of time to establish a diagnosis, the inexperience of the physician, or shortcomings in the patient–physician communication of symptoms, the pathology is often misdiagnosed or the severity of the disease is underestimated, leading to inappropriate, unnecessary, or imprecise therapies, which can result in complex damages to patients’ health (Evans & Johnston, 2011). Migraines can be misdiagnosed as tension headache, sinus headache, or other types of headache. A diagnosis of migraine should be considered when there are recurrent and debiliting headaches without secondary warning signs (Burch et al., 2018; Burch, 2019; Wang et al., 2019). Therefore, misdiagnosis or incorrect classification of the type of migraine and inadequate treatment of the pathology constitute the underlying problem associated with migraines (Deza, 2010; Trillos, 2010).\n\nThis article seeks to contribute to the early identification of different types of migraines through the use of supervised learning techniques based on artificial intelligence, which allow overcoming the difficulties encountered. The purpose of this study is to develop a classification model that allows the determination of the type of migraine a patient suffers based on the analysis of its symptoms and medical history. The novelty, significance, and relevance of this study are outlined as follows:\n\n• An indirect method aimed at classifying the type of migraine experienced by a patient, which, unlike existing methodologies, does not use procedures requiring brain wave measurement or the use of sensors.\n\n• The systematic migraine classification process used includes the stages of data collection based on symptoms and diagnosis by the treating physician, selection of the most relevant features, use of different classification models, and selection of the most suitable model based on the accuracy and precision of diagnosis.\n\n\nMethods\n\nThe strategy developed here is based on a systemic approach oriented to the specification of neural network models for the classification of migraines with and without aura, highlighting the relevance of considering key aspects that lead to strong implications in their application, such as the selection of variables and the performance measures that allow for the selection of the best model.\n\nStarting from the existence of a data source that includes various typical symptoms of patients with migraines, Figure 1 outlines the steps in the classification of patients with migraines and the comparison model (García-González et al., 2019; Sánchez-Sánchez et al., 2019b).\n\nThe elements included in Figure 1 are discussed below.\n\n1. Selection: The data selection phase is directed toward the preliminary analysis of the various data sources (if any), their features, and aspects related to the environment in which they are obtained. This selection can be defined as a process of approximation to the available information through subjective analysis and statistical treatment in order to infer the hidden structure of data.\n\nKnowing the goals and the data that will enable this process are key factors for a successful selection process.\n\n2. Processing: Data processing consists of analyzing and transforming the input variables with the aim of minimizing noise, highlighting important relationships, and detecting errors to enable the recognition of hidden patterns. Processing comprises four types of processes: the first aimed at minimizing noise via the transformation of the object data and the elimination of irregular patterns (atypical data; poor typing; blank, incomplete, and inconsistent data, etc.); the second aimed at scaling the large-sized object data; the third aimed at considering the syntactic transformations of the object data and facilitating its handling, without leading to changes in the results; and the fourth aimed at the selection of the variables, characteristics, or attributes that will be taken into account. This selection largely depends on the knowledge that the data modeler has on the data sources, and it is his/her task to decide whether to include each variable in the model following some previously established criteria. Typically, not all potential variables are equally informative as they may be correlated, present noise, or have no meaningful relationship with the classification.\n\nThe importance of making an adequate selection lies in the difficulties of convergence in learning, which can involve the inclusion of irrelevant variables and poor performance of models. Hota & Shrivas (2014), and Londoño & Sánchez (2015) define the selection of variables as an optimization process intended to identify the best subset of variables from a fixed variable set. The goal of selection is to reduce the size of the input data to facilitate processing and analysis, discarding data that does not further contribute to the subsequent classification process. This saves time in data processing without disregarding the generation of optimal results.\n\nThe selection of variables not only deals with the decrease in cardinality, i.e., setting a partial or predefined limit to the number of attributes that can be considered when creating a model, but also allows attributes to be properly discarded based on their utility for a good analysis process.\n\n3. Classification: Classification involves searching patterns of interest that express dependency on the data and allows groups with similar features to be established. The process essentially consists of assigning each individual (entity or data) its own category or class, thereby creating sets of individuals sharing some feature that differentiate them from the rest. Classes can be binary, Yes or No, or multiclass, which include more than two categories. At this stage, machine learning, whose objective is to develop techniques that allow computers to learn, is used.\n\nThe past few years have seen the proliferation of different automatic classification techniques based in learning machine, including neural networks, decision trees, logistic regression, Bayesian classifiers, nearest neighbor, support vector machines (SVMs), and multiple discriminant analysis (Doupe et al., 2019; Waring et al., 2020).\n\nIn this article, because of the robustness of the data management technique, adaptability, and acknowledged generalization capacity, neural networks are used to classify patients with migraines.\n\nLogistic regression models, SVMs, nearest neighbor, and decision trees are also implemented in order to compare data collected using other classification techniques.\n\n4. Interpretation: In this phase, evaluation of the quality of the model is performed by analyzing and comparing the results from different metrics used for the classification based on the data obtained in the classification stage, which is aimed at understanding the main characteristics of the model.\n\nFor classification problems, common performance metrics are as follows:\n\n▪ Accuracy: Proportion of correctly classified instances.\n\n▪ Precision: Also called positive predictive value, it represents the fraction of correctly predicted positives among those classified as positive.\n\nHowever, the need for tools aimed at enabling appropriate decision-making has led to an accelerated interest in the development of data classification models in recent decades, which is especially intended to overcome the theoretical, conceptual, and practical limitations of many of the techniques currently available. This has resulted in the emergence of a wide range of models, among which neural networks have demonstrated high potential given their adaptability, generalizability, and learning capabilities and because of the possibility of representing nonlinear relationships (Sánchez-Sánchez & García-González, 2017).\n\nSome aspects that justify and favor the development of neural network models for data classification are as follows (Sánchez-Sánchez et al., 2019a):\n\n1. The data generating process is often unknown and difficult to identify, thereby limiting the capacity of parametric models to appropriately classify data. Neural networks are self-adaptive models that do not require a priori assumptions about the problem under study, a highly desirable feature in cases in which the data generating mechanism is unknown (Qi & Zhang, 2001).\n\n2. Real data often show unstable behavior. The ability of the neural network to learn and be generalized allows the model to learn complex behaviors directly from the data and correctly infer the unseen part of the data from the acquired knowledge (De Gooijer & Kumar, 1992).\n\n3. The relationships between the data and the variables that explain its behavior are complex. The universal approximation characteristics of neural networks allow them to identify hidden dependencies, especially nonlinear dependencies (Cybenko, 1989; Franses & Van Dijk, 2000; Hornik, 1991; Hornik et al., 1989), thereby favoring the representation of complex relationships.\n\n4. Data units can be very large or very small. Neural networks are flexible in relation with the values they receive and deliver and do not require prior treatment.\n\n5. Data are obtained from multiple fields of knowledge. Their functional flexibility and characteristics as universal approximators allow neural networks to represent complex behaviors regardless of the field of knowledge those data belong to.\n\nNeural networks and their recent architectures, such as deep neural networks, have been used effectively in data classification tasks and display better results than other techniques, such as logistic regression, decision trees, Bayesian classifiers, etc.; their strength lies in their high capacity to dynamically create complex prediction functions and emulate human learning (Nikam, 2015; Sánchez-Sánchez & García-González, 2017; Sánchez-Sánchez & García-González, 2018).\n\nA neural network is represented as a three-layer model: an input layer, one or more hidden layers, and an output layer (Figure 2) (Sánchez-Sánchez et al., 2020b). An input layer represents the variables that influence the model, hidden layers perform the processing, and the output layer corresponds to the various migraine classes.\n\nMethod: We used the proposed methodology for the classification of migraines.\n\nPopulation: This study used a database comprising 400 medical records of users diagnosed with various pathologies associated with migraines in the research of the master's thesis “Analysis of Artificial Neural Network Models, for a Migraine Diagnosis System with Aura and without Aura” (De la Hoz et al., 2014). Data were recorded by trained medical personnel at the Hospital Materno Infantil de Soledad during the first quarter of 2013. The compiled database contains physician identification, symptoms, diagnosis, and treatment. No patient identifiable data was required. See underlying data (Sanchez-Sanchez et al., 2020a).\n\nProcedure: Selection and processing: The compiled database contains information regarding patient identification, healthcare provider identification, treating physician identification, symptoms, diagnosis, and treatment. Based on these data, tasks related to noise elimination, error detection, and data translation into numerical variables were performed.\n\nFinally, variables that influence the identification of the type of migraine were selected, focusing on symptoms and diagnosis and disregarding identification and treatment variables. This led to a selection of 23 variables associated with the symptoms or signs that a patient may present and 1 variable associated with the diagnosis that allows the identification of the type of migraine. Table 1 presents a list of the 24 identified variables and their description.\n\nThe type of variable states the different values that it can assume and can be either continuous, e.g., age, or binary, e.g., nausea. The variable “Type” indicates the diagnosis issued by the treating physician based on the symptoms and medical record of the patient, with the possibility of presenting of one of the following classifications:\n\n1. Typical aura with migraine\n\n2. Migraine without aura\n\n3. Typical aura without migraine\n\n4. Familial hemiplegic migraine\n\n5. Sporadic hemiplegic migraine\n\n6. Basilar-type aura\n\n7. Other\n\nFigure 3 presents the distribution of cases according to the classification of migraine types carried out in the study and which serves as a criterion for verifying accuracy and precision.\n\nTests are conducted with the complete dataset (23 variables), and variable reduction, recursive elimination of variables, and selection of the best number through cross-validation is applied, the latter in order to eliminate variables with redundant information. This produces a reduced set of 18 variables.\n\nClassification: Preprocessed data are used as inputs to five different classification models: a multilayer perceptron-type neural network (MLP), which is validated using different network configurations that differ in the number of neurons and hidden layers; a logistic regression model; an SVM model; a nearest neighbor model; and an optimized classification and regression tree (CART).\n\nPython 3 script is used with Scikit-learn 0.23.11 and pandas’ 1.0.4 libraries (Sanchez-Sanchez et al., 2020a).\n\nThe neural network model used is equivalent to an MLP trained using backpropagation. The configuration parameters used for the neural network models are as follows:\n\n• Input neurons: 23 variables corresponding to the first 23 variables presented in Table 1 for the complete model and 18 variables for the reduced model.\n\n• Hidden neurons: An iterative optimization process is performed on each hidden layer, varying from 1 to 25. The number of neurons per layer is based on the best accuracy obtained.\n\n• Hidden layers: An incremental construction process of 1 to 4 layers is conducted. The best number of neurons from the previous hidden layer is taken and is iteratively increased from 1 to 25 in the new hidden layer.\n\n• Output neurons: Seven neurons that correspond to each class of migraine\n\n• Transfer function: Logistics\n\n• Performance metrics: Accuracy and precision\n\n• Learning algorithm: Adam2\n\n• Number of epochs: 2000\n\nLogistic regression uses logistic regression regularization with built-in cross-validation, which automatically selects the best hyperparameters for model fit. A 10-fold and random restart model was used3.\n\nThe SVM model uses the SVC function for classification, in which the fit time quadratically scales with the number of samples4.\n\nIn the nearest neighbor model, the optimal number of neighbors is estimated based on the accuracy, and the Euclidean function with uniform weights is used as a distance measure.\n\nThe decision tree model included in the Scikit-learn library corresponds to an optimized version of CART (Classification and Regression Trees) that uses discrete numerical variables and where an iterative process is used to fit the number of levels based on accuracy.\n\nIn all cases, the dataset is divided into two sets: training and testing, the first corresponding to 80% of the data (320) and the second to 20% (80).\n\n\nResults and discussion\n\nResults were validated based on the type of migraine diagnosed by the treating physician and using the respective measurement made during the classification process provided by the neural network.\n\nTable 2 presents the results of the performance measures for various neural network configurations and classification models. Precision, a metric calculated independently for each of the classes, is taken as the weighted average of the seven migraine classes.\n\nThe results presented in Table 2 indicate the following results:\n\n• The maximum accuracy for both 23 and 18 variables is obtained by using a neural network model with 10 hidden neurons, which results in an accuracy of 97.5%. This means that the classification of migraines by the neural network coincides with that issued by the treating physician in 97% of the 80 cases comprising the test set.\n\n• The neural network models show accuracies and precisions >90%, highlighting values obtained with the neural network model with 10 hidden neurons and a hidden layer for 23 variables and 20 hidden neurons for 18 variables, which reaches values >97% in both metrics, thereby indicating adequate classification.\n\n• Those models with all variables have accuracies >80%, with the exception of the nearest neighbor model, which strongly demonstrates that the predicted values coincide with the real values, allowing for the correct classification of the different types of migraine in percentages >80%.\n\n• The maximum average weighted precision was obtained with the neural network model with a layer of 20 hidden neurons that was reduced to 18 variables, which obtained a value of 98%; this indicates that there is a 98% probability that the model classifies the migraine within a certain type and that the treating physician has also classified it as such.\n\n• The precisions obtained using logistic regression and SVM models do not differ greatly from the value obtained using neural networks, even exceeding them in complex neural network configurations with three and four hidden layers.\n\n• Logistic regression, nearest neighbor, and decision tree models show better accuracy values when using models reduced to 18 variables.\n\n• The values of accuracy and precision obtained using neural network models do not favor the increase of hidden layers, leading to reduction phenomena in both metrics as the number of hidden layers and neurons increases, which is representative of overlearning processes.\n\nTo assess the performance of the classification obtained by the best model proposed here, the performance was compared with that from previous studies. Table 3 presents the migraine classification results from previous studies using precision as a performance measure. The proposed neural network model with 10 neurons presents better values than those obtained from the previous studies, with a precision of 97% with all variables and 98% with reduction to 18 variables.\n\nLAD - least absolute deviations, ANN – artificial neural network, SVM – support vector machine, CBR – case-based reasoning\n\n\nConclusions\n\nThis study presents the development of a methodology for migraine classification using artificial neural network models. The results show that neural networks can achieve higher precision and accuracy than other classification models commonly used in machine learning, which is consistent with the results found when compared with various models proposed in the literature. The first experiments included 24 variables involved in migraine diagnosis, achieving a 97% precision level for the neural network model. However, a second testing phase reduced the set of variables to 18, reaching a precision of 98%. This not only proves that the neural network model is effective for the proper classification of the different types of migraine but shows that it can also be improved by considering a reduced set of variables that significantly affect the classification.\n\nThe implementation of migraine classification through neural networks is a powerful tool whose potential has only incipiently been developed and which constitutes a valuable preliminary progress on the broad problem that automatic detection of migraines can encompass. The significance of this work lies in proposing an accurate and timely method of migraine classification that may support the diagnosis established by the treating physician based on an appropriate reading and identification of the primary and secondary symptoms that the patient presents and that results in the appropriate choice of treatment.\n\nThe main novelties are as follows:\n\n• The development of a holistic methodology for migraine classification that encompasses selection of correct data and interpretation of the results obtained.\n\n• The successful use of artificial neural network models for the classification of different types of migraines based on patients’ symptoms, which use a database with data from patients who have experienced migraines and have been diagnosed by their treating physicians, resulting in a model that allows for the near-perfect distinction among the different types of migraine.\n\nIn addition, increasing the number of patient records in the database can lead to more accurate results in migraine classification because it enhances learning.\n\n\nData availability\n\nCode Ocean: Migraine Classification Model. https://doi.org/10.24433/CO.2826453.v1 (Sanchez-Sanchez et al., 2020a)\n\nThis project contains the following underlying data:\n\n- Migraine.cvs (Dataset contain medical records of patient with migraines)\n\n- Migraine Dataset Description.txt (Description of data.)\n\nZenodo: STARD checklist for ‘Automatic Migraine Classification Using Artificial Neural Networks’\n\nhttp://doi.org/10.5281/zenodo.3872279 (Sanchez-Sanchez, 2020)\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\nCode for the model is available from Code Ocean: https://doi.org/10.24433/CO.2826453.v1 (code.ipynb)\n\nLicense: GNU General Public License (GPL)\n\n\nEthical considerations\n\nData used in the recent study are the result of the master's thesis work of the author Juan Manuel Rúa Áscar, who had authorization for their academic use by the Hospital Materno Infantil de Soledad, in accordance with Colombian Law 1581 of 2012 and Decree 1377 of 2013 art. 10, which regulates the treatment of personal information and allows the use for scientific purposes, and that authorization is it extends for the current study.\n\nEthics approval for the use of the original data in the current study was obtained from Universidad Simón Bolívar Research Ethics Committee on 12 May 2020 (reference PRO-CEI-USB-CE-0328-00)\n\n\nConsent\n\nThis research is not a clinical trial, and doesn’t involve any direct patient contact. Anonymized retrospective data collected as part of routine clinical care are included. As a retrospective patient records study, consent was not requested from individual patients. In such cases the ICO code of practice states that explicit consent is not generally required.", "appendix": "Acknowledgments\n\nThe authors express a posthumous recognition to Dr. Manuel Sánchez Rojas for his help in completing this article and his permanent availability to answer our questions and requirements.\n\n\nFootnotes\n\n1Scikit-learn (formerly scikits.learn and also known as sklearn) is a free software machine learning library for the Python programming language. https://scikit-learn.org\n\n2arXiv:1412.6980\n\n3https://scikit-learn.org/stable/modules/linear_model.html#logistic-regression\n\n4https://scikit-learn.org/stable/modules/generated/sklearn.svm.SVC.html#sklearn.svm.SVC\n\n\nReferences\n\nAlkan A, Akben SB: Use of K-means clustering in migraine detection by using EEG records under flash stimulation. Int J Phys Sci. 2011; 6(4): 641–650. 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(eds) Advanced Computing, Networking and Informatics. Smart Innovation, Systems and Technologies 27. Springer, Cham. 2014; 1: 205–211. Publisher Full Text\n\nIsaza R, Cardona E, Celis J: Cefalea: guías de práctica clínica basadas en la evidencia. Ed ASCOFAME. 1997; 42. Publisher Full Text\n\nJackowski K, Jankowski D, Simic D, et al.: Migraine diagnosis support system based on classifier ensemble. In: ICT Innovations. 2014; 329–339. 2014, Springer. Publisher Full Text\n\nKarsan N, Goadsby PJ: Biological insights from the premonitory symptoms of migraine. Nat Rev Neurol. 2018; 14(12): 699–710. PubMed Abstract \| Publisher Full Text\n\nKatsarava Z, Buse DC, Manack AN, et al.: Defining the differences between episodic migraine and chronic migraine. Curr Pain Headache Rep. 2012; 16(1): 86–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKelman L: The triggers or precipitants of the acute migraine attack. Cephalalgia. 2007; 27(5): 394–402. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "66038", "date": "06 Jul 2020", "name": "Shengyuan Yu", "expertise": [ "Reviewer Expertise Headache" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 carefully read and thoroughly considered your manuscript, \"Automatic migraine classification using artificial neural networks\".\nOur comments:\nThis is a meaningful idea to study the migraine classification by artificial neural networks. There are several significant problems with the description of the study and interpretation of the results.\nFirst, the preface of the article is too wordy. We need not to describe the details of migraine as a reviews, including the epidemiology, diagnosis,differential diagnosis and treatment. In the preface, we should include the brief introduction of migraine and artificial neural networks,the current status of research on this subject, the problem in this field, why we do this study.\nSecond, the methods of study should include the Inclusion and exclusion criteria of the study. the reviews of classification progression of migraine should not included in this part,you can put it partial to the discussion.\n\nThird, the discussion is too simple, we advice to discuss the present methods that using in the migraine classification and where is our comparative advantage, potential mechanism of the artificial neural networks using in the migraine classification. By the way, you can points out the direction of future research.\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": "5711", "date": "13 Jul 2020", "name": "Paola Sanchez-Sanchez", "role": "Author Response", "response": "We appreciate the evaluation, which is very timely and pertinent. In response, and following the recommendations, a new version of the manuscript will be made" }, { "c_id": "5722", "date": "17 Jul 2020", "name": "Paola Sanchez-Sanchez", "role": "Author Response", "response": "We sincerely appreciate the reviewer feedback on this work and have improved the article based on your recommendations. We have addressed each comment as follows in the article: 1. We rewrite the preface focusing the content on aspects relevant to research such as a short introduction to migraine, the current state of research in the area, the problems surrounding the classification of different types of migraine, how artificial neural networks help to solve the problems and why of this study. 2. The methods section is rewritten for better clarity in the writing. Clarifications are made regarding the inclusion and exclusion criteria, which are described in the results. 3. The discussion section was expanded to include the validation of the results with the different models applied, the significance of the results obtained, an account of some aspects that justify the development of classification models with artificial neural networks and the comparison with some studies previous. Likewise, the conclusions was expanded to include future research fields." } ] } ]	1	https://f1000research.com/articles/9-618
https://f1000research.com/articles/9-288/v1	24 Apr 20	{ "type": "Software Tool Article", "title": "MetaboMAPS: Pathway sharing and multi-omics data visualization in metabolic context", "authors": [ "Julia Koblitz", "Dietmar Schomburg", "Meina Neumann-Schaal", "Julia Koblitz", "Dietmar Schomburg" ], "abstract": "Metabolic pathways are an important part of systems biology research since they illustrate complex interactions between metabolites, enzymes, and regulators. Pathway maps are drawn to elucidate metabolism or to set data in a metabolic context. We present MetaboMAPS, a web-based platform to visualize numerical data on individual metabolic pathway maps. Metabolic maps can be stored, distributed and downloaded in SVG-format. MetaboMAPS was designed for users without computational background and supports pathway sharing without strict conventions. In addition to existing applications that established standards for well-studied pathways, MetaboMAPS offers a niche for individual, customized pathways beyond common knowledge, supporting ongoing research by creating publication-ready visualizations of experimental data.", "keywords": [ "Systems Biology", "Metabolic Maps", "Pathways", "SVG", "Metabolism", "Data Visualization", "Omics Data" ], "content": "Introduction\n\nThe field of systems biology is based on the integration of data from different biological fields, e.g. transcriptomics, proteomics, metabolomics, modelling, to gain a detailed understanding of an organism. However, the data integration is still challenging to date. In particular, correlating transcriptome or proteome data to metabolome data requires careful revision based on expert knowledge of metabolic pathways and intensive manual work (Cavill et al., 2016). Therefore, easily accessible tools are needed to help during analysis and interpretation of multi-omics data. When one tries to understand metabolic changes, pathway maps are often used for guidance (Cavill et al., 2016). However, the number of pathway maps in scientific publications is large, as is the diversity. Solely the TCA cycle was drawn hundreds of times, being one of the most conserved pathways among all domains of life. However, even such conserved pathways exhibit differences among species: the gut pathogen Clostridioides difficile uses an incomplete TCA cycle (Dannheim et al., 2017) and some Cyanobacteria use a TCA cycle with an additional GABA shunt and a variety of anaplerotic reactions (Will et al., 2019). Conclusively, there are pathway maps that can be used for a broad range of different organisms while others are exclusive for a few species. For this reason, single comprehensive pathway maps, e.g. the KEGG maps, the BRENDA pathway maps, or printed pathway maps (Michal & Schomburg, 2013), have their limitations: the maps cannot provide organism- or group-specific modifications. Moreover, a general map will not contain pathways that are exclusive for small groups of organisms or are currently incompletely understood. For visualization of multi-omics data, a specific map is required both, regarding the organism and the underlying scientific question. Here we present MetaboMAPS (Helmecke, 2020), a novel web-based tool that on one hand, serves as a platform to share metabolic pathway maps in an organism-dependent manner. On the other hand, MetaboMAPS assists during interpretation of metabolism-associated data by visualizing experimental data sets on pathway maps.\n\n\nMethods\n\nPHP is used to access an internal SQL database and to handle file and user management. In addition, a user-friendly web interface is integrated to handle pathway exploration and user interactions. The pathways can be uploaded, stored and downloaded in SVG format. SVG manipulation, including zoom, editing, and plotting of data, is done with the JavaScript Library D3. Hosting, infrastructure maintenance, and issue tracking is provided by the enzyme database BRENDA.\n\nMetaboMAPS (Helmecke, 2020) can be accessed with every modern browser. Log-in is required for upload, editing, and sharing of pathways, but not for exploring pathways, data visualization and downloads.\n\n\nResults\n\nMetaboMAPS (Helmecke, 2020) is a platform where users can upload individual metabolic pathways and release them for the scientific community. In this process, the pathway gets a unique accession number for reference in publications. Furthermore, the user can link pathways to publications. If the pathway map includes unpublished information, it can be uploaded in confidential mode. In this way, the pathway can be shared with specified colleagues and used for data visualization, but is not available for the general public. Pathways can be found by searching category, name, assigned identifier (e.g. EC number, locus tag), or accession. A unique feature of MetaboMAPS is that uploaded maps must not follow strict conventions as other tools require. The style, detail level and content of the maps is according to the scientist’s needs, and since the maps can be downloaded and modified, they can also be adjusted by other users. Pathway rating and the possibility to add comments increase the quality of uploaded pathways via community contributions. In this way, MetaboMAPS does not compete with but complements curated, comprehensive maps that are already well established. It offers a niche for tentative, novel or incomplete pathways to support ongoing research beyond common knowledge. Since MetaboMAPS creates reproducible, customizable visualizations of high quality, it is suitable to generate publication-ready figures with little effort.\n\nEach pathway is associated with one or more organisms. In fact, it is possible to add the same pathway to hundreds of different organisms. An organism overview shows all pathway maps that are associated to a selected organism. On the other hand, the pathway overview page displays all background information, such as a list of authors, the pathway description, links to publications, and all organisms that are associated to this pathway. The pathways and information are also easily accessible on mobile devices.\n\nUsers can upload their own metabolic pathways in SVG format. We chose this particular format because it can be displayed in every modern browser, can be easily manipulated, is completely scalable, and of small file size. Additionally, SVG-files can be exported from every program that users eventually use to draw a metabolic pathway (e.g. Inkscape, Adobe Illustrator, Microsoft Powerpoint, LibreOffice Impress) and users can continue to work with their preferred software.\n\nA unique and highly useful feature of MetaboMAPS is the possibility to visualize experimental data on metabolic pathways. Suitable data sets include but are not limited to transcriptomic, proteomic, metabolomic studies, flux distributions, 13C-flux measurements and others. The process for sharing pathway maps and using them for visualization is shown in Figure 1. The first step is the upload of an existing metabolic pathway in SVG format (Figure 1A). Afterwards, the user can add further information and assign the pathway to a pathway category. In the second step, an intuitive online editor is used to draw plot boxes (Figure 1B), which define the positions where the experimental data should be visualized. Each plot box can be assigned to one or more identifiers, either organism-specific (e.g. locus tags, GIs) or general (e.g. EC numbers, metabolite names). Data from the BKMS (Lang et al., 2011) and BRENDA (Jeske et al., 2019) databases are used to provide auto-completion of metabolites and enzymes, synonym matching, and cross-linking identifiers to other databases, e.g. BRENDA, KEGG, and MetaCyc. The identifier connects a row in the uploaded data set to a specific plot box. In the third step, any type of numerical data can be loaded in the browser and is visualized in the respective plot box (Figure 1C). Data must be in CSV-format, containing the identifiers that connect the data to plot boxes in the first row. Different types of visualization, like colour scales, a number of plot types (e.g. bar charts, line charts, heat maps), and other visual settings offer a high degree of customization. In the end, the pathway including the data visualization as well as legends can be downloaded in SVG or PNG-format.\n\n(A) Upload the metabolic pathway in SVG format. Alternatively, you can use an existing pathway. (B) Draw plot boxes for metabolite (dashed border) and reaction (solid border) associated data. Assign identifiers to each plot box (e.g. EC numbers, locus tags, GI, metabolite synonyms, database IDs). (C) Load your own data set (as CSV file) and visualize reaction and metabolite dependent data simultaneously.\n\n\nConclusion\n\nIn summary, MetaboMAPS (Helmecke, 2020) is a platform for sharing metabolic pathway maps and visualizing data in a metabolic context. It encourages scientists to share individual pathway maps without strict conventions and offers customizable and reproducible visualizations of experimental data. It will grow in collaboration with the community and by further development by the BRENDA team.\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\nSoftware available from: https://metabomaps.brenda-enzymes.org.\n\nSource code available from: https://github.com/JuliaHelmecke/MetaboMAPS.\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.3742817 (Helmecke, 2020).\n\nLicense: GNU General Public License v3.0 or later.", "appendix": "Acknowledgements\n\nWe are grateful to Sabine Eva Will, Tobias Ludwig, Carsten Reuse, and Jacqueline Wolf for excessive beta testing and useful feedback. We thank the BRENDA team for supporting this project and for helpful discussions.\n\n\nReferences\n\nCavill R, Jennen D, Kleinjans J, et al.: Transcriptomic and metabolomic data integration. Brief Bioinform. 2016; 17(5): 891–901. PubMed Abstract \| Publisher Full Text\n\nDannheim H, Will SE, Schomburg D, et al.: Clostridioides difficile 630Δerm in silico and in vivo - quantitative growth and extensive polysaccharide secretion. FEBS Open Bio. 2017; 7(4): 602–15. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHelmecke J: MetaboMAPS: Pathway Sharing and Multi-omics Data Visualization in Metabolic Context (Version 1.1.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3742817\n\nJeske L, Placzek S, Schomburg I, et al.: BRENDA in 2019: a European ELIXIR core data resource. Nucleic Acids Res. 2019; 47(D1): D542–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLang M, Stelzer M, Schomburg D: BKM-react, an integrated biochemical reaction database. BMC Biochem. 2011; 12: 42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMichal G, Schomburg D: Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology: Second Edition.2012. Publisher Full Text\n\nWill SE, Henke P, Boedeker C, et al.: Day and Night: Metabolic Profiles and Evolutionary Relationships of Six Axenic Non-Marine Cyanobacteria. Katz LA (ed.). Genome Biol Evol. 2019; 11(1): 270–94. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "63026", "date": "28 May 2020", "name": "Rachel Cavill", "expertise": [ "Reviewer Expertise multi-omics integration", "pathway analysis", "omics data visualisation." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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, through this short descriptive paper, present MetaboMaps, a new tool for uploading pathways and overlaying data from multiple omics. The pathways are visualised clearly with multiple options for the type of overlays available.\n\nThe program is open source and publicly available, making it easy for others to access and examine as needed.\nThis paper would be strengthened by including a better review of other platforms/tools which do similar or related tasks. In particular, the authors should look at wikipathways, and the combination of wikipathways with pathvisio or cytoscape, and explore how their approach differs from what is currently available through these tools. By highlighting the differences and similarities between approaches it will make it easier for potential users to evaluate the platform and select the best tool for their datasets.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5652", "date": "17 Jul 2020", "name": "Julia Koblitz", "role": "Author Response", "response": "Thank you for your feedback regarding our manuscript. We uploaded a new version of our paper and included a review on other resources as suggested." } ] }, { "id": "63432", "date": "10 Jun 2020", "name": "Bianca H. Habermann", "expertise": [ "Reviewer Expertise Computational Biology", "Systems Biology", "Data Integration" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMetaboMAPS is an interesting small application for making available and displaying numerical data on non-standard pathways. It can be seen as complementing the well-known and standard pathway resources, such as KEGG and others. The advancement of MetaboMAPS is the possibility to store and present incomplete or so far not described pathways. Moreover, it has a nice visualization toolbox to display numerical data on the pathways.\n\nThere are some points that should be addressed:\n\nThe way the introduction is formulated currently indicates that well-established pathway resources are ignorant concerning species-specific differences of pathways. This is not true. KEGG for instance offers a rich selection of organisms and indicates that genes are missing/added to the classical pathway. It is true however when it comes to the overall display of the pathway map, which is – I think – what the authors want to express here. They should reformulate this to reflect what they mean to say properly.\n\nI do not fully understand, why it is necessary to point out the disadvantages of printed pathway maps (in a book, citation Michal & Schomburg) – which by default cannot be used for any form of computerized work?\n\nSparse information is given – also on the website – of how to upload user-provided pathways. For instance, it seems necessary to add an organism, before adding a pathway. This is very un-intuitively presented at the website and should be improved.\n\nThe SVG format offers many advantages. However, it is not so easy to e.g. build on an existing pathway from other resources, as they often do not offer download in SVG format. Do the authors have any thoughts on how the interoperability with existing pathway resources could be improved, e.g. by allowing more and different formats for upload?\n\nShould the resource become more widely used for pathways that are incomplete, it will become a problem that users don’t stick to the same nomenclature. How do the authors think about consolidating novel pathways coming from multiple users that illustrate the same biological pathway, yet under a different name?\n\nWhether or not it is an advantage to offer such great flexibility with respect to the pathway map is questionable. Often, users don’t sick to official gene symbols, but use synonyms or other, officially not recognized gene names. The authors should comment on how they could address this issue of unconventional gene names or rarely used synonyms.\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": "5653", "date": "17 Jul 2020", "name": "Julia Koblitz", "role": "Author Response", "response": "We highly appreciate your feedback regarding MetaboMAPS and the quality of our paper. We uploaded a new version of the latter to address your concerns. Point 1 and 2: We changed to the manuscript accordingly. Point 3: Thank you for your helpful feedback regarding the upload of pathway maps. We reimplemented the upload procedure to be more intuitive and user friendly. Basically, the user can now define which organisms are associated after uploading a pathway. Furthermore, we added a submenu to the navigation bar. Point 4: Interoperability is an important topic. The SVG format was our favourite choice because it can be exported from nearly every tool that users eventually use to draw their pathways. Additionally, there exists a number of conversion tools to convert nearly every vector-based format to SVG (we provide How-Tos on the website). Also existing platforms often provide export to SVG, e.g. we tried to add pathways exported from WikiPathways and BRENDA and it worked perfectly fine. However, we are open to include other data formats (e.g. KGML) in the future. Therefore, we would like to rely on user surveys once the tool has a larger community. Point 5: Regarding concerns about pathway nomenclature, we agree that this will be an issue in the future. At this point, we group pathways roughly using 19 pathway categories. Pathways can furthermore be found using identifiers such as EC numbers or metabolites using the search function. However, with increasing numbers of pathways, we will consider implementing a system based on pathway ontologies (e.g. https://www.ebi.ac.uk/ols/ontologies/pw) at a later time point. Point 6: We agree that flexibility is associated with high variability. For this reason, we implemented a system underlying the identifiers that gives the most possible consistency and connectivity, but maintains the flexibility: among inofficial gene names and locus tags, EC numbers are used for reactions and linked to the BRENDA enzyme database. Furthermore, a synonym database is used to match all known metabolite synonyms and connect them to other databases, i.e. BRENDA, KEGG, MetaCyc, and Sabio-RK." } ] } ]	1	https://f1000research.com/articles/9-288
https://f1000research.com/articles/9-285/v1	23 Apr 20	{ "type": "Brief Report", "title": "Expected immune recognition of COVID-19 virus by memory from earlier infections with common coronaviruses in a large part of the world population", "authors": [ "Johannes M. Dijkstra", "Keiichiro Hashimoto", "Keiichiro Hashimoto" ], "abstract": "SARS-CoV-2 is the coronavirus agent of the COVID-19 pandemic causing high mortalities. In contrast, the widely spread human coronaviruses OC43, HKU1, 229E, and NL63 tend to cause only mild symptoms. The present study shows, by in silico analysis, that these common human viruses are expected to induce immune memory against SARS-CoV-2 by sharing protein fragments (antigen epitopes) for presentation to the immune system by MHC class I. A list of such epitopes is provided. The number of these epitopes and the prevalence of the common coronaviruses suggest that a large part of the world population has some degree of specific immunity against SARS-CoV-2 already, even without having been infected by that virus. For inducing protection, booster vaccinations enhancing existing immunity are less demanding than primary vaccinations against new antigens. Therefore, for the discussion on vaccination strategies against COVID-19, the available immune memory against related viruses should be part of the consideration.", "keywords": [ "Coronavirus", "COVID-19", "SARS-CoV-2", "OC43", "HKU1", "229E", "NL63", "MHC class I", "Immunology", "Vaccination" ], "content": "Introduction\n\nFrom the end of 2019, the world experienced the coronavirus disease 2019 (COVID-19) pandemic caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; aka 2019 novel coronavirus or 2019-nCoV). SARS-CoV-2 shares ~80% nucleotide identity with SARS-CoV-1 (aka SARS-CoV), the causative agent of the SARS epidemy from 2002, and is even more similar to some coronaviruses in bats (Andersen et al., 2020; Ceraolo & Giorgi, 2020; Wu et al., 2020; Zhou et al., 2020). Coronaviruses are membrane-enveloped positive-strand RNA viruses with, for an RNA virus, a large genome of ~30 kb. That genome encodes several structural components of the virion including the nucleocapsid protein N and the membrane proteins S (spike), M, and E, plus also a number of nonstructural proteins involved in RNA replication and other—partly unknown—functions (Weiss & Navas-Martin, 2005). The coronaviruses infecting humans belong to the serological/phylogenetic clades group I (alphacoronaviruses) and group II (betacoronaviruses); group I includes HCoV-229E (human coronavirus 229E) and HCoV-NL63, while group II includes SARS-CoV-1, SARS-CoV-2, Middle East respiratory syndrome coronavirus (MERS-CoV), HCoV-OC43, and HCoV-HKU1. The viruses SARS-CoV-1 and MERS-CoV, on average, cause the most severe symptoms, and their outbreaks were successfully monitored and halted. At the other end of the spectrum, the viruses HCoV-229E, HCoV-NL-63, HCoV-OC43, and HCoV-HKU1 tend to cause only mild symptoms and are very common.\n\nThe Centers for Disease Control and Prevention (CDC; https://www.cdc.gov/coronavirus/general-information.html) states: “Common human coronaviruses, including types 229E, NL63, OC43, and HKU1, usually cause mild to moderate upper-respiratory tract illnesses, like the common cold. Most people get infected with one or more of these viruses at some point in their lives.” The same agency lists the common symptoms caused by these viruses as runny nose, sore throat, headache, fever, cough, and general feeling of being unwell, but also explains that they occasionally cause lower-respiratory tract illnesses, such as pneumonia or bronchitis. The viruses 229E and OC43 have been known since the 1960s (reviewed in Kahn & McIntosh, 2005), but NL63 (van der Hoek et al., 2004) and HKU1 (Woo et al., 2005) were only (conclusively) identified following the rise in interest in coronaviruses in the wake of the SARS epidemy. These common coronaviruses are believed to be the second most common cause of the common cold (Mäkelä et al., 1998). In the U.S.A., a 3-year RT-PCR surveillance of respiratory samples of patients revealed that the four viruses 229E, NL63, OC43, and HKU1 were present at levels varying by season and region, with all individual viruses peaking at >3% prevalence in each investigated region (Midwest, Northeast, South, West); co-infection with other coronaviruses was found in only ~2% of infected cases, but co-infection with another respiratory virus was found in a substantial ~30% of infected cases (Killerby et al., 2018). This pattern was reminiscent of findings in the United Kingdom (Gaunt et al., 2010) and Japan (Matoba et al., 2015). Serological investigations in countries as diverse as the U.S.A. (Bradburne & Somerset, 1972; Dijkman et al., 2012), China (Zhou et al., 2013), and Qatar (Al Kahlout et al., 2019), found that most healthy blood donors had antibodies against coronaviruses, supporting that these viruses are widespread indeed.\n\nSince immune memory protection can be induced by related pathogens, as exemplified by the eradication of human smallpox virus (Variola) by immunization with a related “cowpox” virus (Vaccinia) (Plotkin & Plotkin, 2018), it is interesting to consider whether common human coronavirus infections may have induced some level of protection against SARS-CoV-2.\n\nThe two major arms of immune memory concern antibody secretion by B cells and killing of infected cells by CD8+ T cells. For a coronavirus infection in mouse, both immune responses were needed to efficiently control the virus (reviewed by Weiss & Navas-Martin, 2005). Based on theoretical considerations alone, it is difficult to predict effective B cell memory across different virus species (Qiu et al., 2020), and very recent experiments concluded that sera from people that likely had been infected with the common human coronaviruses 229E, NL63, OC43, and/or HKU1, possessed no or negligible cross-reactivity with SARS-CoV-2 virus S protein (Amanat et al., 2020) and thus probably possess no neutralizing antibodies. However, for inducing CD8+ T cell memory, the core requirement is merely that an identical peptide is presented by major histocompatibility complex (MHC) class I (MHC-I) molecules. MHC-I molecules present peptide fragments from intracellular proteins, thus also from viral proteins, at the cell surface for screening by CD8+ cytotoxic T cells (Neefjes et al., 2011). CD8+ T cells recognize the combination of MHC-I molecule with peptide by T cell receptors (TCR) that are unique per T cell clone, and if stimulated these clones can proliferate, kill the presenting (virus-infected) cell, and produce memory cells. MHC-I molecules are polymorphic in that they are represented by many diverse allelic forms that differ between human populations and individuals (Robinson et al., 2020), and mostly bind 9 amino acids (aa) length in their binding groove which is closed at either end (Bjorkman et al., 1987; Rammensee et al., 1995; Schellens et al., 2015).\n\nIn the present study, we analyzed whether there are linear 9 aa epitopes that are identical between proteins encoded by SARS-CoV-2 and one or more of the common human coronaviruses. We found many of such epitopes indeed, and, by using prediction software, found that some are expected to bind well to certain MHC-I alleles. We therefore expect that common human coronaviruses can induce some level of CD8+ T cell-mediated immune memory recognizing SARS-CoV-2, and consider the possibility of enhancing that immune memory by vaccination.\n\n\nMethods\n\nProteins encoded by a reported genomic sequence for SARS-CoV-2 (GenBank MN908947; Wu et al., 2020) were compared with those for HCoV-OC43 (NC_005147; Vijgen et al., 2005), HCoV-HKU1 (NC_006577; Woo et al., 2005), HCoV-229E (NC_002645; Thiel et al., 2001), and HCoV-NL63 (NC_005831; van der Hoek et al., 2004) by performing BLAST homology searches at the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and by making multiple sequence alignments using CLUSTALW software (https://www.genome.jp/tools-bin/clustalw); continuous stretches of 9 aa acids identical between SARS-CoV-2 and one of the other viruses were identified manually. All these shared 9 aa epitopes were screened by ANN 4.0 software at IEDB Analysis Resource (http://tools.immuneepitope.org/mhci/) for prediction of their affinity to a set of representative human MHC-I alleles.\n\n\nResults and discussion\n\nTable 1 lists the 9 aa epitopes that are identical between proteins encoded by SARS-CoV-2 and one or more of the common human coronaviruses. Many identical >9 aa stretches were found with ORF1ab encoded polyprotein, one such identical stretch (of 12 aa) was found with the N protein of the other two type II coronaviruses HCoV-OC43 and HCoV-HKU1, and no such stretches were found when comparing with any of the other gene products; ORF1ab-derived mature proteins with such stretches, expected from cleavage of the polyprotein precursor (Wu et al., 2020), were the transmembrane protein nonstructural protein 4 (NSP4), 3C-like cysteine protease NSP5, RNA binding protein NSP9, RNA dependent RNA polymerase NSP12, helicase NSP13, 3’-to-5’ exonuclease NSP14, nidoviral endoribonuclease specific for U NSP15, and S-adenosylmethionine-dependent ribose 2’-O-methyltransferase NSP16 (Table 1). Sequence alignment figures of the ORF1ab and N proteins are shown in Extended data (Dijkstra, 2020) with highlighting of the interesting epitopes. It is of note that the S protein, which is the prime candidate for inducing neutralizing antibodies (Cohen, 2020), is not suitable for inducing an MHC-I-restricted immune memory across the investigated viral species as between S protein of SARS-CoV-2 and S proteins of the common human coronaviruses there are no 9 aa or even 8 aa matches (not shown).\n\nCompared sequences were derived from the following GenBank accessions: for ORF1ab SARS-CoV-2, QHD43415; OC43, NP_937947; HKU1, YP_173236; 229E, NP_073549; NL63, YP_003766; and for N SARS-CoV-2, QHD43423; OC43, NP_937954; HKU1, YP_173242; 229E, NP_073556; NL63, YP_003771 Source positions indicate the N-terminal position of the depicted 9 aa sequence in the SARS-CoV-2 ORF1ab protein or N protein (see Supplementary file 1). The ORF1ab protein is only a precursor polyprotein, and the column Mature protein indicates the probable mature protein that possesses the epitope: 3CLpro, 3C-like cysteine protease; RdRp, RNA dependent RNA polymerase; Hel, helicase; ExoN, 3’-to-5’ exonuclease; NendoU, nidoviral endoribonuclease specific for U; O-MT, S-adenosylmethionine-dependent ribose 2’-O-methyltransferase. Yellow blocks indicate the presence of identical sequences in the respective common human coronavirus, and gray blocks indicate the absence of such matches. Orange blocks highlight those peptides with predicted IC50 values of <500 nM for one of the twelf investigated MHC-I alleles.\n\nTable 1 shows that there are >200 linear epitopes of 9 aa that are identical between SARS-CoV-2 and at least one of the common human coronaviruses, most of them with OC43 and HKU1 which, like SARS-CoV-2, belong to the group II coronaviruses. In a simplified model, if people would have been exposed to many of these epitopes through common HCoV infections, this kind of equals immunization by a small intracellular protein under natural viral infection conditions. Whereas live virus is commonly considered the gold standard in regard to inducing strong immunity, unless the virus has some tricks up its sleeve to manipulate the immune system, which for common human coronaviruses is not well investigated, a research grant proposal suggesting this as a vaccination strategy would probably fail. Reviewers of such proposal would righteously point out that the strategy would not induce neutralizing antibodies, which for combating some viral infections can be very important, and that for inducing MHC-I-restricted cell-mediated cytotoxicity memory, ideally, a much larger protein or more proteins should be taken. Those reviewers would conclude that for such small intracellular protein to induce strong immune memory it would need too much luck in regard to immunogenicity and it would be too dependent on the MHC alleles of the immunized person as different alleles bind different peptides. Nevertheless, those reviewers would probably also agree that in most persons thus vaccinated some (small) level of immune memory protection would be established, even with such small non-surface protein (e.g. Polakos et al., 2001). Regardless of that this obviously is not the ideal way to induce a population-wide strong protective immunity (see the spread of COVID-19), together with other factors such as health and the number of encountered viruses (the strength of the viral challenge), the induced immune memory could make a difference for whether a person gets sick; at the population scale, it so may somewhat reduce the virus reproduction number. Importantly, by stimulating this HCoV-derived MHC-I restricted immune memory by vaccination (see below), it could become a more significant helper in fighting COVID-19.\n\nBased on combinations of experimental results and computer learning, various software has been created that with some degree of reliability can predict how efficiently peptides can bind to the grooves of various MHC-I alleles. In the present study we used the artificial neural network (ANN) function (Lundegaard et al., 2008) of the IEDB Analysis Resource (http://tools.immuneepitope.org/mhci/) (Dhanda et al., 2019) which may achieve >75% reliability for predicting binding (Lundegaard et al., 2008). The software owners state that IC50 values of <50 nM and <500 nM are considered high and intermediate affinity, respectively, and are found for most epitopes known to stimulate cytotoxic T cells. Therefore, Table 1 only indicates the predicted IC50 values if lower than 500 nM. Table 1 shows these expected affinities for twelve MHC-I alleles that are rather representative for sets of MHC-I alleles with similar binding properties (supertypes) and so represent a large part of the human MHC-I binding repertoire (Lund et al., 2004): HLA-A0101 (supertype A1), HLA-A0201 (A2), HLA-A0301 (A3), HLA-A2402 (A24), HLA-A2601(A26), HLA-B0702 (B7), HLA-B0801 (B8), HLA-B1501 (B62), HLA-B2705 (B27), HLA-B3901 (B39), HLA-B4001 (B44), and HLA-B5801 (B58). It is of note that Li et al. (2008) found that a SARS-CoV-1 15 aa peptide sequence (their “Replicase 4701-4715” peptide) encompassing the SARS-CoV-2/HCoV-shared ORF1ab4725 and ORF1ab4726 epitopes that are predicted to bind well to the MHC-I alleles HLA-A0201 and HLA-B3901 (see our Table 1) was associated with a CD8+ T cell response against SARS-CoV-1 in humans. However, Li et al. (2008) also found such CD8+ T cell response associated with a SARS-CoV-1 15 aa peptide (their “Nucleocapsid 106-120” peptide) encompassing the SARS-CoV-2/HCoV-shared N 106, N 107, N 108, an N 109 epitopes for which our analyses did not predict MHC-I binding (see our Table 1).\n\nThe MHC-I binding affinity is considered the most selective in determining which peptides are presented, but also steps in the peptide processing and loading pathways may play selective roles which are difficult to capture in prediction software (Nielsen et al., 2005). We argue that, if such steps would be selective for presentation, in most cases they would probably not differentiate between the 9 aa epitope in the SARS-CoV-2 context versus the respective HCoV context, since most of those epitopes are within stretches that also show many similarities in the neighboring residues (Extended data).\n\nNot all stable complexes of MHC-I with non-self peptides elicit a strong immune response, but “immunogenicity” features are hard to predict with meaningful reliability by in silico analysis (Calis et al., 2013), and in the present study we refrain from such predictions. Table 1 should, foremost, be understood as evidence of principle and a list of promising peptides, whereas only future experiments can prove MHC-I-mediated immune memory involving these or other peptides.\n\nIn regard to SARS-CoV-2 recognition, the common human coronaviruses may also induce some MHC-II-mediated immune memory by CD4+ helper T cells (for shared epitope use by different coronaviruses see Zhao et al., 2016). CD4+ helper T cells can help stimulate cells involved in antibody or cell-mediated cytotoxic immune responses (Neefjes et al., 2011). However, for this topic we refrained from detailed (software) predictions because comparison of MHC-II epitopes across different viruses is harder than for MHC-I epitopes. Namely, although the core of MHC-II bound peptides is also only 9 aa, the surrounding amino acids are also part of the bound peptide that tends to be 12-25 aa (Brown et al., 1993; Rammensee et al., 1995; Stern & Wiley, 1994) and can affect how the peptide interacts with the receptors on the CD4+ helper T cells (Arnold et al., 2002).\n\nImmune memory means that a secondary immune response, upon renewed encounter with the same pathogen, is faster and stronger than the primary immune response during the first encounter with the pathogen. This is based on expansion of specific B and T cell clones, which specifically recognize pathogen(-derived) epitopes, with some of those cells becoming memory cells (Paul, 2013). This principle also causes that for a booster vaccination/immunization the requirements for efficiently inducing an immune response are lower than for a first vaccination/immunization (e.g. Goding, 1996). Especially in elderly people, who have a decreased ability to mount adaptive immune responses against new antigens, vaccination that stimulates an immune memory response may be beneficial (Reber et al., 2012). As discussed above, people’s past infections with common coronaviruses probably did not induce a B cell memory for making antibodies that can neutralize SARS-CoV-2. However, as the current study shows by analysis of linear 9 aa epitopes, these common human coronaviruses are expected to induce CD8+ T cells that may potentially kill SARS-CoV-2-infected cells and so can help eradicate the virus. There are several possible ways to exploit this probable immune memory. For example, if using RNA for immunization (Cohen, 2020), it may be best to also include SARS-CoV-2 genes that encode MHC-I epitopes that match those of the common coronaviruses. Alternatively, delivery of these epitopes to the MHC-I presentation system may be tried by peptide or protein based vaccines (e.g. Kohyama et al., 2009; Slingluff, 2011; van Montfoort et al., 2014; Yadav et al., 2014), possibly in combination with some of the strategies that are currently being explored for non-specific stimulation of the immune system against COVID-19 (Kupferschmidt & Cohen, 2020). Protein (-coding) vaccines, for example encompassing a large part of the SARS-CoV-2 ORF1ab product, would have an advantage over peptide-vaccines by including multiple possible MHC-I and also MHC-II epitopes, and be less dependent on MHC-allele matching and the quality of software predictions. Naturally, as for any new vaccine strategy, it should be carefully assessed whether the benefits of the induced type of immunity outweigh the potential deleterious health effects caused by an increased inflammation response (Cohen, 2020; Weingart et al., 2004). Important questions are also whether the history of previous—especially recent—infections with common coronaviruses, or people’s MHC alleles, affect people’s resistance to SARS-CoV-2. Most definitely, if discussing possible strategies for vaccination against SARS-CoV-2, pre-existing MHC-I-based immunity derived from previous infections with common coronaviruses should be part of the consideration.\n\n\nNotification\n\nAlthough we were not aware of this at the time of writing, a recent preprint appeared with overlapping contents (Nguyen et al., 2020).\n\n\nData availability\n\nSevere acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome, Accession number MN908947: https://www.ncbi.nlm.nih.gov/nuccore/MN908947\n\nHuman coronavirus OC43, complete genome, Accession number NC_005147.1: https://www.ncbi.nlm.nih.gov/nuccore/NC_005147.1?report=genbank\n\nHuman coronavirus HKU1, complete genome, Accession number NC_006577: https://www.ncbi.nlm.nih.gov/nuccore/NC_006577\n\nHuman coronavirus 229E, complete genome, Accession number NC_002645: https://www.ncbi.nlm.nih.gov/nuccore/NC_002645\n\nHuman Coronavirus NL63, complete genome, Accession number NC_005831: https://www.ncbi.nlm.nih.gov/nuccore/NC_005831\n\nHarvard Dataverse: Extended data. Sequence alignments of SARS-CoV-2 ORF1ab and N proteins with their counterparts in the common human coronaviruses, https://doi.org/10.7910/DVN/CNPUPA (Dijkstra, 2020).\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "References\n\nAl Kahlout RA, Nasrallah GK, Farag EA, et al.: Comparative Serological Study for the Prevalence of Anti-MERS Coronavirus Antibodies in High- and Low-Risk Groups in Qatar. J Immunol Res. 2019; 2019: 1386740. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmanat F, Nguyen T, Chromikova V, et al.: A serological assay to detect SARS-CoV-2 seroconversion in humans. medRxiv. 2020. Publisher Full Text\n\nAndersen KG, Rambaut A, Lipkin WI, et al.: The proximal origin of SARS-CoV-2. Nat Med. 2020; 26(4): 450–452. 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J Virol. 2004; 78(22): 12672–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeiss SR, Navas-Martin S: Coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. Microbiol Mol Biol Rev. 2005; 69(4): 635–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWoo PC, Lau SK, Chu CM, et al.: Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia. J Virol. 2005; 79(2): 884–95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu F, Zhao S, Yu B, et al.: A new coronavirus associated with human respiratory disease in China. Nature. 2020; 579(7798): 265–269. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYadav M, Jhunjhunwala S, Phung QT, et al.: Predicting immunogenic tumour mutations by combining mass spectrometry and exome sequencing. Nature. 2014; 515(7528): 572–6. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "63655", "date": "22 Jun 2020", "name": "Anna Gil", "expertise": [ "Reviewer Expertise T cel viral immunology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript reports a very useful study that extends our knowledge of peptide-MHC recognition by CD8+ T cells during emerging virus infections such as SARS-CoV-2. Detailed in silico analysis showed the presence of potential epitopes shared between new types of betacoronavirus: SARS-CoV-2 and common human alphacoronaviruses: OC43, HKU1, 229E and NL63. Due to the high prevalence of the common coronaviruses authors suggest that the large part of the human population has already some degree of specific memory T cell response before having been infected with the virus.\nAs authors already mentioned in their manuscript the similar study by Nguyen A. et al (JVI, 20201) demonstrated the HLA binding affinity of all possible 8- to 12-mers from SARS-CoV-2 proteome. This group found that HLA-B15:03 type has the greatest capacity to present highly conserved peptides which are shared among coronaviruses suggesting a cross-protective T cell immune response. In current manuscript using different prediction software authors identified and showed the sequence of epitopes which bind well to similar HLA type, HLA-B15:01. Interestingly, one of the epitopes (YLRKHFSM) can be bound by 4 different HLA types. The obvious strength of this study is the demonstration that certain epitopes, which are identical between SARS-CoV-2 and the common human coronaviruses are being predicted as high affinity binders in multiple HLA-A and B types.\nOverall, the work reports important new details about SARS-CoV-2 epitopes theoretically being recognized by human CD8+ T cells. Undeniably, future experiments can prove if generated memory immune responses are specific to the proposed epitopes.\nThere are some suggestions:\nThe analysis of p/MHCI binding for HLA-C type (if available) would certainly complete the list of presented epitopes\n\nThe introduction part subtitled: ”The possibility of matching linear epitopes…” has missing information about previously published reports regarding T cell response in individuals infected with coronaviruses, either common or SARS-CoV.\n\nIn the discussion part readers may wonder why the authors did not discuss their findings with those already published (although they may not have been out at the time of submission) but should be included in the revision.\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? 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": "5696", "date": "17 Jul 2020", "name": "Johannes M. Dijkstra", "role": "Author Response", "response": "Dear Dr. Gil and Dr. Selin, Thank you for your kindness to review our article. We are glad that you find our study very useful, and that you agree with the conclusions. Coming from experts like you, this is very reassuring. We have now added a bit more information on the Nguyen et al. study in the Notification section. The advantage of the Nguyen et al. study was that they were more complete on SARS-CoV-2 MHC epitope predictions and made an association with MHC allele distributions. The advantage of our paper is a more concentrated focus on the memory expected from previous coronavirus infections, and the vaccination potential deriving from that memory. They were first, which we acknowledged, although we wrote our paper independent of their article, and during the submission process of our article theirs was not an indexed publication yet. Presumably, this type of overlap will happen a lot with a topic as intensively studied as coronavirus, and we feel we took a reasonable approach for dealing with their study. From your reviews, we understand that you and the other reviewer, Dr. Sant, find this within acceptability. Thank you for referring to the interesting YLRKHFSMMIL stretch which is identical between HCoV-NL63 and SARS-CoV-2, as indeed it harbors predicted binding epitopes for several MHC-I supertypes. However, we prefer not to discuss this in text form, because there are many uncertainties (e.g., about recent HCoV-NL63 distributions in the world population) and a textual discussion may not add clarity to the table presentation. Based on your advice, we now have added HLA-C predictions to Table 1. Likewise, we now have added a more extensive summary of previous reports on T cell memory after coronavirus infections. Apart from addressing the reviewer’s comments, we corrected a mistake and now informed the readers that there is a single 8 aa match between compared S proteins. Apart from addressing the reviewer’s comments, we now also added the information that the study by Callow et al. (1990) on HCoV-229E, concluded imperfect immune memory protection even by live virus infection one year before challenge. Again, we like to thank you for the reviewing, as we are aware of the time and effort that it takes. We are very happy that our article is appreciated, since it deals with such an important topic. Sincerely, also on behalf of Keiichiro Hashimoto Hans (J.M.) Dijkstra" } ] }, { "id": "63246", "date": "22 Jun 2020", "name": "Andrea J. Sant", "expertise": [ "Reviewer Expertise Immunology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThese analyses of potential cross reactive CD8 T cell epitopes between the current SARS-CoV-2 and “seasonal” endemic human CoV is useful and timely and the discussion is balanced.\n\nThere are several modifications that I believe would improve the clarify and value of the manuscript.\n\nBased on the first sentence of the paragraph entitled “the possibility of matching linear epitopes…”, the authors sate that the two major arms of immune memory…are antibody and CD8 T cells” I believe this is incorrect, as CD4 T cells can directly impact lung pathology and contribute to both protective and pathological immune responses. In fact a recent paper uploaded to BioRxiv suggested that it was populations in the CD4 T cell compartments that correlated with disease severity. The authors should acknowledge that all three subsets of the adaptive response (B cells, CD8 and CD4 T cells) are likely to be important, but this manuscript focusses on CD8 epitopes.\n\nThe authors refer to the “software owners” when describing cutoffs. They are perhaps better described as software “designers”.\n\nWhen discussing “Vaccine Potential”, the authors state that the secondary response is “faster and stronger”. This should be more accurately described, with some references, in a way that points out the higher frequency of responding cells during memory recall, and lower thresholds of TcR engagement needed for T cell activation, both qualities that contribute to a competitive advantage of memory cells.\n\nBecause the nature of CD8 memory to the different antigens screened by the authors is not known, the epitopes identified may or may not be targets of cross reactive memory recall. Therefore, the word “expected” should be substituted for “Potential” or some other word that indicates that the epitope list includes candidates but not expected epitopes.\nI think the Table could be made quite a lot smaller and thus more valuable to the reader. The source proteins could be indicated as an abbreviation provided in the legend as could the various seasonal strains. The boxes could then be quite small, and either be positive or negative. In any case, an effort should be made to condense this table.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5697", "date": "17 Jul 2020", "name": "Johannes M. Dijkstra", "role": "Author Response", "response": "Dear Dr. Sant, Thank you for reviewing our article. We highly appreciate your comment that our article is timely and well balanced. Especially the latter is important, since we did not want to make a general audience too enthusiastic, while we simultaneously wanted to stress that there is a chance that this MHC-I-mediated immunity might (possibly after boostering by vaccines) give real protection. You are correct that immune memory by CD4+ cells is not only relevant to their control of B cell and CD8 T cell responses, and we have rewritten the sentence and paragraph on the “major arms of immune memory.” As you state, our paper focusses on CD8+ T cell memory indeed, because that is the memory response that can be predicted most reliably by sequence analysis alone. In the notification section we now shortly discuss two papers that appeared after we submitted our paper, and which claim to have found SARS-CoV-2 recognizing CD4+ and CD8+ T cell memory derived from previous common coronavirus infections. As requested, we have now changed “software owners” to “software designers.” As requested, we now have added more references of an enhanced immune reaction after a second (booster) immunization. However, we do not feel that for our type of paper it is necessary to discuss the mechanisms of memory T cells besides just mentioning their involvement. As for the words “expected” versus “potential.” We feel that we used the word “expected” correctly. Table 1, where all the peptides are listed, carries neither of these two words and has a very neutral title. In the text, some peptides are referred to as “expected” to bind a particular MHC molecule, a term clearly relating to the indicated software and literature. Given the large number of potential MHC-I epitopes shared between the viruses, we “expect” previous common coronavirus infections to have induced some CD8+ T cell immune memory that recognizes SARS-CoV-2; this claim is not about the protective value of this memory, and we feel, therefore, that the word “expect” is within reasonability. As for the Table format. The format was chosen by the journal editorial team, and we can see that for some uses it has advantages. However, we understand your concern, and now have added an Excel format variant of the Table to the supplement section so that readers can more easily view and interact with the data. Apart from addressing the reviewer’s comments, we corrected a mistake and now informed the readers that there is a single 8 aa match between compared S proteins. Apart from addressing the reviewer’s comments, we now also added the information that the study by Callow et al. (1990) on HCoV-229E, concluded imperfect immune memory protection even by live virus infection one year before challenge. We are aware of the time and effort reviewing takes, and are very thankful of your thoughtful contribution. It lifts our spirits that you and the other reviewers consider our article a nice contribution to the COVID-19 studies. Sincerely, also on behalf of Keiichiro Hashimoto Hans (J.M.) Dijkstra" } ] } ]	1	https://f1000research.com/articles/9-285
https://f1000research.com/articles/9-716/v1	17 Jul 20	{ "type": "Research Article", "title": "Evaluation of antioxidant and anti-collagenase activity of Rosa damascena L. flower petal and receptacle extract", "authors": [ "Elfitriani Elfitriani", "Ahmad Raif", "Chrismis N. Ginting", "Refi Ikhtiari", "Elfitriani Elfitriani", "Ahmad Raif", "Chrismis N. Ginting" ], "abstract": "Background: Rosa damascena L. is the most notable species of the Rosaceae family in the world, and has been used in food, cosmetics, and the pharmaceutical industry. Bioactive compounds in this flower are known to have several activities, such as antioxidant, antimicrobial, and anti-inflammatory. In this study, the antioxidant and collagenase inhibitory activities of R. damascena L. petal and receptacle extracts were evaluated. Methods: Ethanolic extraction of R. damascena L. petals (EERP) and R. damascena L. receptacles (EERR) were obtained, and bioactive compounds (flavonoids, phenolics, alkaloids, steroids, tannins, terpenoids, and triterpenoids) were classified by phytochemical screening. Antioxidant activities were analyzed by Ferric Reducing Antioxidant Power (FRAP) assay, while anti-collagenase analysis was examined through the inhibition of collagenase. Results: Phytochemical test revealed the presence of flavonoids, phenolics, alkaloids, steroids, triterpenoids, triterpenes, and tannin. EERP showed higher FRAP activity (164.23 ± 1.34 μM Fe(II)) than EERR (12.85 ± 6.19 μM Fe(II)). EERP also had higher inhibitory activity of collagenase (IC50 = 115.48±1.78 µM/mL) compared to EERR (IC50 = 141.96±6.13 µM/mL). Conclusions: R. damascena L. petal and receptacle ethanol extracts contain several components, such as phenolics, flavonoids, alkaloids, tannins, terpenes, triterpenoids, and steroids. These extracts exhibit antioxidant activity and collagenase inhibition. R. damascena L. petal extract showed higher antioxidant activity through FRAP assay and inhibitory activity of collagenase than R. damascena L. receptacle extract.", "keywords": [ "Rosa damascena L.", "Ferric Reducing Antioxidant Power", "antioxidant", "anti-collagenase" ], "content": "Introduction\n\nAntioxidants are compounds that can react with Reactive Oxygen Species (ROS) to prevent the oxidation of lipids, DNA, and proteins1. Several studies report that ROS correlates with several human diseases, such as cancer2, inflammation3, and the aging process4. In the aging process, ROS play a role in protein unfolding, lipid breakdown, DNA damage, fragmentation, and collagen disintegration5. In the body, ROS promote the activation of collagenase and block collagen synthesis, leading to a decrease in skin elasticity and acceleration of skin wrinkles. This mechanism by ROS is the major factor of degeneration collagen fiber6.\n\nRosa damascena L. is the most important species among the Rosaceae family of flowers. This species has many pharmacological properties and has been used in food, herbal medicine, and cosmetics since ancient times7. Previous research showed that R. damascena L. has anti-viral, anti-microbial, anti-cancer, anti-depressant, anti-inflammatory, anti-convulsant, and anti-oxidant activities8. Several studies report that phenolic compounds and flavonoids contributed to an increase in antioxidant activity7,9–11. In this study, we evaluate the antioxidant and anti-collagenase activity from ethanol extract of R. damascena L. petals and receptacles.\n\n\nMethods\n\nR. damascena L. flowers were obtained from Bandung, Indonesia (-6.803662, 107.586633). In total, 1400 g and 700 g of petals and receptacles of R. damascena L. were dried and mashed, respectively. The dried samples (250 g petals and 90g receptacles) were soaked with 8000 mL and 3000 mL of 70% ethanol, respectively, and macerated. This solvent was replaced every 3 days and the extracted solution from the first to the last (15th day) was mixed and concentrated using a rotary evaporator at 50°C for 4 h. The extracts were labeled as ethanol extract of rose petals (EERP) and ethanol extract of rose receptacles (EERR).\n\nPhytochemical analysis was done to detect the presence of flavonoids, phenolics, tannins, steroids/triterpenoids, terpenoids and alkaloids. 10 mg of extracts were used for each analysis following the procedures of Widowati et al. without modification12.\n\nThe antioxidant activity of EERP and EERR was investigated using FRAP assay following Sricharoen et al. with slight modifications13. Briefly, ferric chloride hexahydrate (Merck) and 2,4,6-tripyridyl-s-triazine (TPTZ) (Sigma) were mixed to form the FRAP reagent. 142.5 μL FRAP solution was added into 7.5 μL of sample extract and the mixtures were incubated at 37°C for 5 min. The absorbance of the reaction mixture was read at 539 nm. A set of concentrations 0–200 µg/mL of FeSO4.7H2O (Sigma) was used for calibration of the standard curve. The results were expressed as μM Fe(II)/μg extract.\n\nAnti-collagenase activity of EERR and EERP was assessed by evaluating the inhibition of collagenase, following the procedure of Wittenuer et al.14 10 µL collagenase from Clostridium histolyticum (0.01 U/mL, Sigma), 60 µL of 50 mM Tricine buffer pH 7.5 (containing 10 mM CaCl2 (Merck) and 400 mM NaCl (TCL Co., Ltd)), and 30 µL of extract (7.81 – 250 µg/mL) were mixed and incubated at 37°C for 20 min. After incubation, 20 μL of 1 mM N-[3-(2- Furyl)acryloyl]-leu-gly-Pro-Ala (FALGPA, Sigma) in Tricine buffer (Sigma) was added to the mixture. The collagenase inhibitory activities were measured at 335 nm by monitoring for 20 min after starting the reaction. The inhibition value was calculated according to Eq. (1). IC50 values were determined from dose-effect curves.\n\n\n\nStatistical analysis using SPSS version 23 was carried out by normality test using numeric variable with Levene test and ANOVA and continued with post-hoc honestly significant difference, and Tukey test to the confidence level of 95% (α = 0.05). All data are expressed as mean ± SEM.\n\n\nResults and Discussion\n\nPhytochemical investigations of R. damascena L. petal and receptacle extracts in ethanol expressed positive results of flavonoids, phenolic compounds, tannins, steroids, terpenoids, and alkaloids. The details are presented in Table 1.\n\nEERP, ethanolic extraction of R. damascena L. petals; EERR, ethanolic extraction of R. damascena L. receptacles.\n\nThe results of phytochemical screening showed steroids are detected in EERP by the bluish-green color, while in the same test, EERR gave a red color indicated it contained triterpenes. Although there was a difference between chemical compounds, both samples contained phenol and flavonoids that act as antioxidants and anti-collagenases. In previous research, Patil et al.15 found that the ethanol extract of R. damascene Mill L. contains total phenolic and flavonoid compounds at a higher level than acetone, aqueous, and chloroform extracts.\n\nThe antioxidant and anti-collagen activity of flowers depends on the compounds it contains and the solvent used. Phenolics are well known to have antioxidant capability9–10,16, as does flavonoids and alkaloids17. Steroids, terpenoids, and triterpenoids also have been reported to have antioxidant activity18. Muccilli et al.19 reported that tannins also play a role in blocking oxidative chain reactions. The solvent extract also has a contribution to increasing antioxidant and anti-collagenase activity. Ethanol extract of R. damascena L. petals has been shown to have a lower IC50 value than acetone when evaluated by DPPH15. Ethanol extracts also show the highest phenolic content and antioxidant activity than water extracts of R. spinosissima9.\n\nTotal antioxidant activity of EERP and EERR were further evaluated by FRAP assay. The FRAP value was obtained by plotting the standard curve of FeSO4 at a concentration between 0.78 and 50 μg/mL (Figure 1).\n\nThe antioxidant activity of both extracts is presented in Figure 2. In this test, the antioxidant activities were obtained based on the capacity to reduce ferric (III) to ferrous (II). The result demonstrates that EERP has higher antioxidant capacity compared to EERR.\n\nEERP, ethanolic extraction of Rosa damascena L. petals; EERR, ethanolic extraction of R. damascena L. receptacles.\n\nTable 2 showed FRAP values for different concentrations of EERP and EERR. The data showed normal distribution and was significantly different (p > 0.005). Both extracts showed higher antioxidant activity with higher concentrations (Figure 2). The highest antioxidant activities were observed at 50 at µg/mL (EERP: 164.23 ± 1.34 µM/mL; EERR: 12.85 ± 6.19 µM/mL).\n\nEERP, ethanolic extraction of Rosa damascena L. petals; EERR, ethanolic extraction of R. damascena L. receptacles.\n\nData presented in the form of mean ± SEM. Different lowercase letters indicate a significant difference in p < 0.05 (Post Hoc Tukey HSD Test).\n\nCollagen is the most abundant protein responsible for conferring skin thickness, strength, and elasticity20. This metalloproteinase enzyme has a vital role in collagen degradation, which increases aging signs21. The percentage of collagenase inhibition of EERP and EERR found in the present study is presented in Table 3. Anti-collagenase activity showed a concentration-dependent manner, where higher concentrations of the extracts increased the anti-collagenase (and thus antiaging) activity of extracts. At the highest concentration (250 µM/mL) EERP showed the highest inhibition activity compared to EERR (91.52 ± 3.44 µg/mL and 77 ± 2.03 µg/mL, respectively).\n\nEERP and EERR were diluted in tricine buffer to reach the final concentration of 7.81; 15.63; 31.25; 62.50; 125.00; 250.00 (µg/mL). EERP, ethanolic extraction of Rosa damascena L. petals; EERR, ethanolic extraction of R. damascena L. receptacles.\n\nData presented in the form of mean ± SEM. Different lowercase letters indicate a significant difference in p < 0.05 (Post Hoc Tukey HSD Test).\n\nThe IC50 percentage can be seen in Table 4. The IC50 value of EERR is higher than EERP (141.96 ± 6.13 µM/mL and 115.48± 1.78 µM/mL, respectively). This indicates that EERR has less activity to impede collagenase activity than EERP; however, these results show that there is potential for EERR to inhibit collagenase. Park et al.22 found that R. damascena extract impacted metalloproteinase transcription by suppressing AP-1 activation. AP-1 is a protein activated by UVB penetration to the skin, and initiates changes of the extracellular matrix, including collagen, elastin, and proteoglycans degradation. Our results show that ethanol extracts of petals and receptacles of R. damascene L. possess anti-collagenase activity.\n\nEERP, ethanolic extraction of Rosa damascena L. petals; EERR, ethanolic extraction of R. damascena L. receptacles.\n\n\nConclusion\n\nR. damascena L. petal and receptacle ethanol extracts contain several components, such as phenolics, flavonoids, alkaloids, tannins, terpenes, triterpenoids, and steroids. The pharmacological effects of these extracts exhibit antioxidant activity and collagenase inhibition. R. damascena L. petal extract showed higher antioxidant activity through FRAP assay and inhibitory activity of collagenase than R. damascena L. receptacle extract.\n\n\nData availability\n\nFigshare: Raw data Analysis of anticollagenase an IC50 value.xlsx, https://doi.org/10.6084/m9.figshare.12585626.v123\n\nFigshare: Raw data FRAP Activity of Extracts of Rosa Damascene Petals and Receptacles, https://doi.org/10.6084/m9.figshare.12585671.v124\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nBast A, Haenen G: Nutritional Antioxidants: It Is Time to Categorise. In Antioxidants in Sport Nutrition. Lamprecht, M., Ed. CRC Press/Taylor & Francis © 2015 by Taylor & Francis Group, LLC.: Boca Raton (FL): 2015. PubMed Abstract\n\nAggarwal V, Tuli HS, Varol A, et al.: Role of Reactive Oxygen Species in Cancer Progression: Molecular Mechanisms and Recent Advancements. Biomolecules. 2019; 9(11): 735. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChelombitko MA: Role of Reactive Oxygen Species in Inflammation: A Minireview. Moscow Univ Biol Sci Bull. 2018; 73(4): 199–202. Publisher Full Text\n\nXu H, Zheng YW, Liu Q, et al.: Reactive Oxygen Species in Skin Repair, Regeneration, Aging, and Inflammation. In Reactive Oxygen Species (ROS) in Living Cells.2018. Publisher Full Text\n\nHasan RR, Jat D: Oxidative stress and antioxidants: an overview. International Journal of Advanced Research and Review. 2017; 2(9): 110–119. Reference Source\n\nChoi EK, Guo H, Choi JK, et al.: Extraction conditions of white rose petals for the inhibition of enzymes related to skin aging. Lab Anim Res. 2015; 31(3): 148–152. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAl-Garni AA, Rahimulddin SA, Doghaither HAA, et al.: Evaluation of the antioxidant activities of aqueous extracts of fresh madeni rose petals. Int J Sci Nat. 2017; 8(3): 461–468. Reference Source\n\nMahboubi M: Rosa damascena as holy ancient herb with novel applications. J Tradit Complement Med. 2015; 6(1): 10–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoczka N, Stefanovits-Bányai É, Ombódi A: Total polyphenol content and antioxidant capacity of rosehips of some rosa species. Medicines (Basel). 2018; 5(3): 84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSengul M, Sener D, Ercisli S: The determination of antioxidant capacities and chemical properties of rosa (Rosa damascena Mill.) products. Acta Sci Pol Hortorum Cultus. 2017; 16(4): 63–72. Publisher Full Text\n\nDjamaan A, Noviza D, Septianingsih D, et al.: The use of purple sweet potato (Ipomoea batatas) starch as binder in mangosteen peel extracts lozenges formulation. Der Pharma Chemica. 2016; 8(2): 410–414. Reference Source\n\nWidowati W, Fauziah N, Herdiman H, et al.: Antioxidant and anti aging assays of Oryza sativa extracts, vanillin and coumaric acid. Journal of Natural Remedies. 2016; 16(3): 88–99. Publisher Full Text\n\nSricharoen P, Techawongstein S, Chanthai S: A high correlation indicating for an evaluation of antioxidant activity and total phenolics content of various chilli varieties. J Food Sci Technol. 2015; 52(12): 8077–8085. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWittenauer J, Mäckle S, Sußmann D, et al.: Inhibitory effects of polyphenols from grape pomace extract on collagenase and elastase activity. Fitoterapia. 2015; 101: 179–187. PubMed Abstract \| Publisher Full Text\n\nPatil PS, Tatke PA, Gabhe SY: In vitro Antioxidant and Free Radical Scavenging Activity of Extracts of Rosa damascena Flower Petals. American Journal of Phytomedicine and Clinical Therapeutics. 2015; 589–601. Reference Source\n\nNayebi N, Khalilib N, Kamalinejad M, et al.: A systematic review of the efficacy and safety of Rosa damascena. Mill. with an overview on its phytopharmacological properties. Complement Ther Med. 2017; 34: 129–140. PubMed Abstract \| Publisher Full Text\n\nJiang XL, Wang L, Wang EJ, et al.: Flavonoid glycosides and alkaloids from the embryos of Nelumbo nucifera seeds and their antioxidant activity. Fitoterapia. 2018; 125: 184–190. PubMed Abstract \| Publisher Full Text\n\nKoduru S, Jimoh FO, Griegson DS, et al.: Antioxidant Activity of Two Steorid Alkaloids Extracted from Solanum aculeastrum. J Pharmacol Toxicol. 2007; 2(2): 160–167. Publisher Full Text\n\nMuccilli V, Cardullo N, Spatafora C, et al.: α-Glucosidase inhibition and antioxidant activity of an oenological commercial tannin. Extraction, fractionation and analysis by HPLC/ESI-MS/MS and 1H NMR. Food Chem. 2017; 215: 50–60. PubMed Abstract \| Publisher Full Text\n\nAvila Rodríguez MI, Rodríguez Barroso LG, Sánchez ML: Collagen: A review on its sources and potential cosmetic applications. J Cosmet Dermatol. 2018; 17(1): 20–26. PubMed Abstract \| Publisher Full Text\n\nAlipour H, Raz A, Zakeri S, et al.: Therapeutic applications of collagenase (metalloproteases): A review. Asian Pac J Trop Biomed. 2016; 6(11): 975–981. Publisher Full Text\n\nPark B, Hwang E, Seo SA, et al.: Dietary Rosa damascena protects against UVB-induced skin aging by improving collagen synthesis via MMPs reduction through alterations of c-Jun and c-Fos and TGF-β1 stimulation mediated smad2/3 and smad7. J Funct Foods. 2017; 36: 480–489. Publisher Full Text\n\nElfitriani E: Raw data Analysis of anticollagenase an IC50 value.xlsx. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12585626.v1\n\nElfitriani E: Raw data FRAP Activity of Extracts of Rosa Damascene Petals and Receptacles. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12585671.v1" }	[ { "id": "86477", "date": "12 Aug 2021", "name": "Ihsan Iswaldi", "expertise": [ "Reviewer Expertise Food Chemistry and Analysis", "Bioactive Ingredients", "Analytical Separation" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 can be accepted in this form after the affirmation of several issues:\nTannins are highly polymerized compounds of polyphenols and flavonoids are also chemically defined families of polyphenols. So, the authors do not need to mention polyphenols in line with flavonoids and tannins because the last two are classified as polyphenols. Unless the authors want to mention the classes of polyphenols, then write them in sequence.\n\nThe authors should explain why they only used FRAP assay for antioxidant activity. Why did not they carry out DPPH, ABTS, TEAC, ORAC, and TBARS methods as well and compare the results?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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": "118942", "date": "11 Jan 2022", "name": "Oksana Sytar", "expertise": [ "Reviewer Expertise Plant secondary metabolites", "especially plant natural compounds with healthy effects" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, The article “Evaluation of antioxidant and anti-collagenase activity of Rosa damascena L. flower petal and receptacle extract” is original research work and can be indexed after major revision.\nPlease, see some comments below:\nIn Introduction part it would be good to add more information about previous studies with representatives family Rosaceae.\n\nIt would be good to admit that research studies were done with identification of flavonoids and phenolics in the leaves of some representatives family Rosaceae but less current studies about petal biochemical composition (e.g. Sytar et al., 20151; Sytar et al., 20182). Also for Rosa damascena was studied well biochemical composition of essential oils but not petal which can be source of essential oil depending from method of extraction (Mahboubi et al., 20113; Piotrowicz et al., 20214).\n\nIn the part material and methods for line “Phytochemical analysis was done to detect the presence of flavonoids, phenolics, tannins, steroids/triterpenoids, terpenoids and alkaloids.” - please add information if it was used spectrophotometrically or other kind of methods.\n\nPlease, admit that evaluation of anti-collagenase activity of Rosa damascena petals was done for the first time. Add information why it is important to study anti-collagenase activity.\n\nIn the part where is written about Phytochemical test results for ethanol extracts of Rosa damascena L. petals and receptacles, please add a sentence that the method of visual assessment was recommended to use for pre-screening of anthocyanins content and rutin for some plants (Sytar et al., 20145).\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/9-716
https://f1000research.com/articles/9-223/v1	01 Apr 20	{ "type": "Software Tool Article", "title": "clustifyr: an R package for automated single-cell RNA sequencing cluster classification", "authors": [ "Rui Fu", "Austin E. Gillen", "Ryan M. Sheridan", "Chengzhe Tian", "Michelle Daya", "Yue Hao", "Jay R. Hesselberth", "Kent A. Riemondy", "Rui Fu", "Austin E. Gillen", "Ryan M. Sheridan", "Chengzhe Tian", "Michelle Daya", "Yue Hao", "Jay R. Hesselberth" ], "abstract": "Assignment of cell types from single-cell RNA sequencing (scRNA-seq) data remains a time-consuming and error-prone process. Current packages for identity assignment use limited types of reference data and often have rigid data structure requirements. We developed the clustifyr R package to leverage several external data types, including gene expression profiles to assign likely cell types using data from scRNA-seq, bulk RNA-seq, microarray expression data, or signature gene lists. We benchmark various parameters of a correlation-based approach and implement gene list enrichment methods. clustifyr is a lightweight and effective cell-type assignment tool developed for compatibility with various scRNA-seq analysis workflows. clustifyr is publicly available at https://github.com/rnabioco/clustifyr", "keywords": [ "Single-cell RNA sequencing", "cell type classification", "gene expression profile", "R package" ], "content": "Introduction\n\nSingle-cell mRNA sequencing (scRNA-seq) promises to deliver elevated understanding of cellular mechanisms, cell heterogeneity within tissue, and developmental transitions1–5. A key challenge in scRNA-seq data analysis is the identification of cell types from single-cell transcriptomes. Manual inspection of the expression patterns from a small number of marker genes is still standard practice, which is cumbersome and frequently inaccurate. Unfortunately, current implementations of scRNA-seq suffer from several limitations3,6,7 that further compound the problem of cell type identification. First, only RNA levels are measured, which may not correlate with cell surface marker or gene expression signatures identified through other experimental techniques. Second, due to the low capture rate of RNAs, low expressing genes may face detection problems regardless of sequencing depth. Many previously established markers of disease or developmental processes suffer from this issue, such as transcription factors. On the data analysis front, over or under-clustering can generate cluster markers that are uninformative for cell type labeling. In addition, cluster markers that are unrecognizable to an investigator may indicate potentially interesting unexpected cell types, but can be very intimidating to interpret.\n\nFor these reasons, investigators struggle to integrate scRNA-seq into their studies due to the challenges of confidently identifying previously characterized or novel cell populations. Formalized data-driven approaches for assigning cell type labels to clusters greatly aid researchers in interrogating scRNA-seq experiments. Currently, multiple cell type assignment packages exist but they are specifically tailored towards input types or workflows8–13. As more and more approaches to the classification problem are introduced, benchmarking performance and compatibility to sequencing platforms and analysis pipelines becomes increasingly important.\n\nWe developed the R package clustifyr, a lightweight and flexible tool that leverages a wide range of prior knowledge of cell types to pinpoint target cells of interest or assign general cell identities to difficult-to-annotate clusters. Here, we demonstrate its basic usage and applications with transcriptomic information of external datasets and/or signature gene profiles, to explore and quantify likely cell types. The clustifyr package is built with compatibility and ease-of-use in mind to support other popular scRNA-seq tools and formats.\n\n\nMethods\n\nclustifyr requires query and reference data in the form of raw or normalized expression matrices, corresponding metadata tables, and a list of variable genes (Figure 1).\n\n\n\nclustifyr adopts correlation-based methods to find reference transcriptomes with the highest similarity to query cluster expression profiles, defaulting to Spearman ranked correlation, with options to use Pearson, Kendall, or Cosine correlation instead if desired. clustify() will return a matrix of correlation coefficients for each cell type and cluster, with the row names corresponding to the query cluster number and column names as the reference cell types.\n\n\n\nQuery clusters are assigned cell types to the highest correlated reference cell type, with an automatic or manual cutoff threshold for query clusters dissimilar to all available reference cell types, to be labeled as “unassigned”.\n\n\n\nTo better integrate with standard workflows that involve S3/S4 R objects, methods for clustifyr are written to directly recognize Seurat14 (v2 and v3) and SingleCellExperiment15 objects, retrieve the required information, and reinsert classification results back into an output object. A more general wrapper is also included for compatibility with other common data structures, and can be easily extended to new object types. This approach also has the added benefit of forgoing certain calculations such as variable gene selection or clustering, which may already be stored within input objects.\n\n\n\nIn the absence of suitable reference data (i.e. RNA-seq or microarray expression matrices), clustifyr can build scRNA-seq reference data by averaging per-cell expression data for each cluster, to generate a transcriptomic snapshot. Direct reference-building from SingleCellExperiment or Seurat objects is supported as well.\n\n\n\nData exploration plotting functions, for dimensional reduction scatter plots and heatmaps, are extended from ggplot2 and ComplexHeatmap packages, featuring colorblind-friendly default colors. Simple gene list-based methods (clustify_lists()) for sanity checks on positive and negative markers, via gene list enrichment or calculation of percentage detection by cluster, are implemented as well.\n\nCorrelation method. We benchmarked clustifyr against a suite of comparable datasets, PBMCbench16, generated from two PBMC samples using multiple scRNA-seq methods. Notably, for each reference dataset cross-referenced to other samples, clustifyr achieved a median F1-score of above 0.94 using Spearman ranked correlation (Figure 2A). Other correlation methods are on par or slightly worse at cross-platform classifications, which is expected based on the nature of ranked vs unranked methods. We therefore selected Spearman as the default method in clustifyr, with other methods also available, as well as a wrapper function to find consensus identities across available correlation methods (call_consensus()).\n\nA) Comparison of accuracy of different correlation methods for classifying across platforms using the PBMCbench dataset. B) Heatmap showing correlation coefficients between query cell types and the reference cell types. Clusters with correlation < 0.50 are assigned as Neg.Cell by clustifyr. C) Comparison of classification power with and without feature selection. D) An assessment of the accuracy of using single or multiple averaged profiles as reference cell types was conducted using the PBMCbench test set. The number of reference expression profiles to generate for each cell type is determined by the number of cells in the cluster (n), and the sub-clustering power argument (x), with the formula nx. E)Accuracy and performance were assessed with decreasing query cluster cell numbers using the PBMCbench test.\n\nCorrelation minimum cutoff. Recognition of missing reference cell types, so as to avoid misclassification, is another point of great interest in the field. From general usage of clustifyr, we find using a minimum correlation cutoff of 0.5 or 0.4 is generally satisfactory. Alternatively, the cutoff threshold can be determined heuristically using 0.8 * highest correlation coefficient among the clusters. One example is shown in Figure 2B, using PBMC rejection benchmark data modified by the SciBet package17. Megakaryocytes were removed from reference data, and labeled as “neg.cells” for ground truth in test data. clustifyr analysis found the “neg.cells” to be dissimilar to all available reference cell types, and hence left as “unassigned” under the default minimum threshold cutoff. Next, we applied clustifyr to a series of increasingly challenging datasets from the scRNAseq_Benchmark13 unseen population rejection test. Without the corresponding cell type references, 57.5% of T cells were rejected and unassigned. When only CD4+ references were removed, 28.2% of test CD4+ T cells were rejected and unassigned. clustifyr was unable to reject CD4+/CD45RO+ memory T cells, mislabeling them as CD4+/CD25 T Reg instead when the exact reference was unavailable. However, these misclassifications are also observed with other classification tools benchmarked in the scRNAseq_Benchmark study13.\n\nVariable gene selection. As the core function of clustifyr is ranked correlation, feature selection to focus on highly variable genes is critical. In Figure 2C, we compare correlation coefficients using all detected genes (>10,000) vs feature selection by the package M3Drop. A basic level of feature selection, e.g. M3Drop, Seurat VST (default takes top 2,000), or simply 1,000 genes with highest variance in the reference data, is sufficient to classify the pancreatic cells. In the case of other cell type mixtures, especially ones without complete knowledge of the expected cell types, clustering and feature selection will be of greater importance. clustifyr does not provide novel clustering or feature selection methods on its own, but instead is built to maintain flexibility to incorporate methods from other, and future, packages. We view these questions as a fast-moving fields18,19, and hope to benefit from new advances, while keeping the general clustifyr framework intact.\n\nSubclustering. For scRNA-seq reference data, matrices are built by averaging per-cell expression data for each cluster, to generate a transcriptomic snapshot similar to bulk RNA-seq or microarray data. An additional argument to subcluster the reference dataset clusters is also available, to generate more than one expression profile per reference cell type. The number of subclusters for each reference cell type is dependent on the number of cells in the cluster (n), and the sub-clustering power argument (x), following the formula nx9. However, this approach does not improve classification in the PBMCbench data (Figure 2D). We envision its utility would greatly depend on the granularity of the clustering in the reference dataset.\n\nCells per cluster. After testing a general reference set built from the Mouse Cell Atlas20 to be of high accuracy in classification of the Tabula Muris data, we subsampled the query data (Figure 2E). As expected, with further downsampling of the number of cells in each query cluster, we observe decreased accuracy. Yet, even at 15 cells per tested cluster, clustifyr still performed well, with a further increase in speed. Based on these results, we set the default parameters in clustifyr to exclude or warn users of classification on clusters containing less than 10 cells. In addition, an intentional overclustering and classification function based on k-means clustering (overcluster_test()) is implemented in clustifyr for exploration of clustering quality.\n\nUsing clustifyr, peripheral blood mononuclear cell (PBMC) clusters from the Seurat PBMC 3k tutorial are correctly labeled using either bulk-RNA seq references generated from the ImmGen database9,21, processed microarray data of purified cell types22, or previously annotated scRNA-seq results from the Seurat CBMC CITE-seq tutorial14 (Figure 3).\n\nUMAP projections showing the ground truth cell types, or cell types called by clustifyr using different data sources (microarray or bulk RNA-seq data from purified cell types, or scRNA-seq data).\n\nTo assess the performance of clustifyr, we used the Tabula Muris dataset5, which contains data generated from 12 matching tissues using both 10x 3’ end seq (“drop”) and SmartSeq2 (“facs”) platforms. Using references built from “facs” Seurat objects, we attempted to assign cell type identities to clusters in “drop” Seurat objects. In benchmarking results, clustifyr is comparably accurate versus other automated classification packages (Figure 4A). Cross-platform comparisons are inherently more difficult, and the approach used by clustifyr is aimed at being platform- and normalization-agnostic. Mean runtime, including both reference building and test data classification, in Tabular Muris classifications was ~ 1 second if the required variable gene list is extracted from the query Seurat object. Alternatively, variable genes can be recalculated by other methods such as M3Drop23, to reach similar results. Correlation-based clustifyr classification performed better than hypergeometric-based gene list enrichment as implemented in clustify_lists.\n\nA) Accuracy and run-time of classifications generated by clustifyr or existing methods using the Tabula Muris to benchmark cell type classifications across sequencing platforms. Each point represents a different tissue comparison. B) Performance comparison of clustifyr to existing methods by subsampling the Tabula Muris dataset. Cell numbers are listed in the facet labels. C) Performance comparison of clustifyr to existing methods testing against Allen Institute Brain Atlas data containing 34 cell types.\n\nFor scalability benchmarking, we adapted scRNAseq_Benchmark subsampling for the Tabula Muris dataset. Once again, clustifyr is accurate and efficient, compared to other developed methods (Figure 4B). We also reached similarly satisfactory results in scRNA-seq brain transcriptome data from mouse and human samples, as detailed by scRNAseq_Benchmark pipeline using data from the Allen Institute Brain Atlas13 (Figure 4C).\n\nclustifyr was tested against scmap v1.8.08, SingleR v1.0.19, Seurat v3.1.114, latest GitHub versions of ACTINN11 and scPred12, and SVM as implemented in python3 scikit-learn v0.19.124. scRNA-seq Tabula Muris data was downloaded as seuratV2 objects. Human pancreas data wasdownloaded as SCE objects. In all instances, to mimic the usage case of clustifyr, clustering and dimension reduction projections are acquired from available metadata, in lieu of new analysis.\n\nAn R script was modified to benchmark clustifyr following the approach and datasets of scRNAseq_Benchmark13, using M3Drop23 variable gene selection for every test. R code used for benchmarking, and preprocessing of other datasets, in the form of matrices and tables, are documented in R scripts available in the clustifyr and clustifyrdata GitHub repositories.\n\nclustifyr is distributed as part of the Bioconductor R package repository and is compatible with Mac OS X, Windows, and major Linux operating systems. Package dependencies and system requirements are documented in the clustifyr Bioconductor repository.\n\n\nConclusions\n\nWe present a flexible and lightweight R package for cluster identity assignment. The tool bridges various forms of prior knowledge and scRNA-seq analysis. Reference sources can include scRNA-seq data with cell types assigned (or average expression per cell type, which can be stored at much smaller file sizes), sorted bulk RNA-seq, and microarray data. clustifyr, with minimal package dependencies, is compatible with a number of standard analysis workflows such as Seurat or Bioconductor, without requiring the user to perform the error-prone process of converting to a new scRNA-seq data structure, and can be easily extended to incorporate other data storage object types. clustifyr is designed to perform classification after previous steps of analysis by other informatics tools. Therefore, it relies on, and is agnostic to, common external packages for cell clustering and variable feature selection. We envision it to be compatible with all current and future scRNA-seq processing, clustering, and marker gene discovery workflows. Benchmarking reveals the package performs well in mapping cluster identity across different scRNA-seq platforms and experimental types. As we and others observe25, novel algorithms may not be necessary for cell type classification, at least within the current limitations of sequencing technology and our broadstroke understanding of cell “types”. Rather, the generation of community curated reference databases is likely to be critical for reproducible annotation of cell types in scRNA-seq datasets.\n\nOn the user end, clustifyr is built with simple out-of-the-box wrapper functions, sensible defaults, yet also extensive options for more experienced users. Instead of building an additional single-cell-specific data structure, or requiring specific scRNA-seq pipeline packages, it simply handles basic data.frames (tables) and matrices (Figure 1). Input query data and reference data are intentionally kept in expression matrix form for maximum flexibility, ease-of-use, and ease-of-interpretation. Also, by operating on predefined clusters, clustifyr has high scalability and minimal resource requirements on large datasets. Using per-cluster expression averages results in rapid classification. However, cell-type annotation accuracy is therefore heavily reliant on appropriate selection of the number of clusters. Users are therefore encouraged to explore cell type annotations derived from multiple clustering settings. Additionally, assigning cell types using discrete clusters may not be appropriate for datasets with continuous cellular transitions such as developmental processes, which are more suited to trajectory inference analysis methods. As an alternative, clustifyr also supports per-cell annotation, however the runtime is greatly increased and the accuracy of the cell type classifications are decreased due to the sparsity of scRNA-seq datasets, and requires a consensus aggregation step across multiple cells to obtain reliable cell type annotations.\n\nTo further improve the user experience, clustifyr provides easy-to-extend implementations to identify and extract data from established scRNA-seq object formats, such as Seurat14, SingleCellExperiment15, URD4, and CellDataSet (Monocle)26. Available in flexible wrapper functions, both reference building and new classification can be directly achieved through scRNA-seq objects at hand, without going through format conversions or manual extraction. The wrappers can also be expanded to other single cell RNA-seq object types, including the HDF5-backed loom objects, as well as other data types generated by CITE-seq and similar experiments27. Tutorials are documented online to help users integrate clustifyr into their workflows with these and other bioinformatics software.\n\n\nSoftware availability\n\nclustifyr is available from Bioconductor:https://bioconductor.org/packages/devel/bioc/html/clustifyr.html\n\nUp-to-date source code, tutorials, and prebuilt references available from: https://github.com/rnabioco/clustifyr\n\nArchived source code as at time of publication:https://doi.org/10.5281/zenodo.371858828\n\nData used in examples and additional prebuilt references available from: https://github.com/rnabioco/clustifyrdata\n\nLicense: MIT\n\n\nData availability\n\nOriginal raw data used in benchmarking is available from the following sources:", "appendix": "Acknowledgements\n\nA previous version of this article is available on bioRxiv: https://doi.org/10.1101/855064.\n\n\nReferences\n\nZheng GX, Terry JM, Belgrader P, et al.: Massively parallel digital transcriptional profiling of single cells. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLun AT, McCarthy DJ, Marioni JC: A step-by-step workflow for low-level analysis of single-cell RNA-seq data with Bioconductor [version 2; peer review: 3 approved, 2 approved with reservations]. F1000Res. 2016; 5: 2122. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDing J, Adiconis X, Simmons SK, et al.: Systematic comparative analysis of single cell RNA-sequencing methods. bioRxiv. 2019; 632216. Publisher Full Text\n\nLi C, Liu B, Kang B, et al.: SciBet: a fast classifier for cell type identification using single cell RNA sequencing data. bioRxiv. 2019; 645358. Publisher Full Text\n\nDuò A, Robinson MD, Soneson C: A systematic performance evaluation of clustering methods for single-cell RNA-seq data [version 2; peer review: 2 approved]. F1000Res. 2018; 7: 1141. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPedregosa F, Varoquaux G, Gramfort A, et al.: Scikit-learn: Machine Learning in Python. J Mach Learn Res. 2011; 12: 2825–30. Reference Source\n\nKöhler ND, Büttner M, Theis FJ: Deep learning does not outperform classical machine learning for cell-type annotation. bioRxiv. 2019 [cited 2020 Jan 28]; 653907. Publisher Full Text\n\nCao J, Spielmann M, Qiu X, et al.: The single-cell transcriptional landscape of mammalian organogenesis. Nature. 2019; 566(7745): 496–502. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRicher AL, Riemondy KA, Hardie L, et al.: Simultaneous measurement of biochemical phenotypes and gene expression in single cells. bioRxiv. 2019; 820233. Publisher Full Text\n\nFu R, Gillen A, Sheridan R, et al.: rnabioco/clustifyr 0.99.7 (Version 0.99.7). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3718588" }	[ { "id": "61913", "date": "20 Apr 2020", "name": "Keegan Korthauer", "expertise": [ "Reviewer Expertise Statistical genomics", "bioinformatics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article introduces a user-friendly and inter-operable R package for cell-type assignment of single-cell RNA-sequencing data. As clearly stated by the authors, the method heavily relies on the (1) results of and (2) any assumptions made by the clustering algorithm applied to the query dataset. The method has potential to be widely useful given its flexibility to take input and give output from many different existing (and future) algorithms. Although the methods proposed are not novel (simple correlation metrics), the software serves to streamline one of the most common procedures in single-cell RNA-sequencing analysis. As detailed below I have some questions regarding the evaluation of the method compared to existing approaches, and a suggestion to more widely distribute the prebuilt references curated as part of the study.\nMajor comments:\nThe 'unseen population rejection test' is an informative measure. However, it is not clear without going back to the scRNAseq_Benchmark (Abdelaal et al., 20191) how clustifyr's performance compares to other tools. It would be useful to give some quantitative or visualization that conveys this comparison.\n\nThe approach is aimed at being \"normalization-agnostic\" as stated in 'Benchmarking' section. However, it's not clear whether this refers to clustifyr in general, or just using the rank correlation setting. If in general, this property should be demonstrated.\n\nThe benchmarking results provided are very helpful, but it's not clear why only a (differing) subset of the methods was applied to each evaluation (i.e. panels of Figure 4 in particular).\n\nMinor comments:\nFrom the description of the method, it seems that if the query dataset is 'over-clustered', meaning a cell-type is incorrectly split into two clusters, clustifyr can return the same cell type assignment for both clusters (provided the correct reference had the highest correlation, and that correlation was above the threshold). Is this correct? If not, please clarify.\n\nThe prebuilt references in the clustifyrdata github repository has potential utility to researchers who don't already have a reference dataset. It might be a good fit to build these reference datasets as a Bioconductor ExperimentHub package.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "5694", "date": "16 Jul 2020", "name": "Kent Riemondy", "role": "Author Response", "response": "We thank the reviewer for their helpful criticisms. Our responses are indicated in italics below. Major comments: The 'unseen population rejection test' is an informative measure. However, it is not clear without going back to the scRNAseq_Benchmark (Abdelaal et al., 20191) how clustifyr's performance compares to other tools. It would be useful to give some quantitative or visualization that conveys this comparison. We agree and have provided an additional figure panel (4E) that provides a visual comparison of clustifyr’s performance compared to tools assessed by the scRNAseq_Benchmark. The approach is aimed at being \"normalization-agnostic\" as stated in 'Benchmarking' section. However, it's not clear whether this refers to clustifyr in general, or just using the rank correlation setting. If in general, this property should be demonstrated. We are referring the property of rank correlation rather than a specific feature of clustifyr. We have amended the text (subsection: Variable gene selection and normalization) to make this point more clear and provide recommendations that users try to implement the same normalization scheme for reference and query data if possible. The benchmarking results provided are very helpful, but it's not clear why only a (differing) subset of the methods was applied to each evaluation (i.e. panels of Figure 4 in particular). We agree that the benchmarking would be more clearly presented by providing more complete assessment of methods across each evaluation. We have updated figures 4A,B,C, and D to consistently present clustifyr’s performance and accuracy compared to other methods. Of note, we were unable to benchmark scPred when examining the Allen Brain Atlas data ( Figure 4C), due to an error that we were unable to troubleshoot. Minor comments: From the description of the method, it seems that if the query dataset is 'over-clustered', meaning a cell-type is incorrectly split into two clusters, clustifyr can return the same cell type assignment for both clusters (provided the correct reference had the highest correlation, and that correlation was above the threshold). Is this correct? If not, please clarify. The reviewer's comment is correct, clustifyr will assign the cell type with the highest correlation, that meets a minimum cut-off value. For over-clustered query cell types, clustifyr will therefore return the same cell-type label, despite the overclustering. Clustifyr also provides a function (overcluster_test()) to intentionally overcluster the query dataset to potentially identify subpopulations that were grouped into another cell type due to inappropriate query dataset clustering. We have included an additional figure panel (2E) to illustrate this functionality. The prebuilt references in the clustifyrdata github repository has potential utility to researchers who don't already have a reference dataset. It might be a good fit to build these reference datasets as a Bioconductor ExperimentHub package. We thank the reviewer for this suggestion and have built an ExperimentHub package that includes the prebuilt references in the clustifyrdata repository. The package (clustifyrdatahub) has been submitted to Bioconductor." } ] }, { "id": "63065", "date": "02 Jun 2020", "name": "Kamil Slowikowski", "expertise": [ "Reviewer Expertise Bioinformatics", "computational biology", "immunogenomics", "scRNA-seq." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 R package for annotating cell clusters in scRNA-seq datasets. Specifically, the package implements code for computing correlations between the columns of two data matrices. They show that high correlations between unknown cell clusters in the first data matrix and annotated cell types in the second matrix can be used to label the unknown cell clusters. They try varying parameters and show the effects on the results, and they also benchmark the time and accuracy compared to other packages designed to annotate scRNA-seq data.\nDetails of the code, methods, and analyses are partly provided. Some details seem to be missing (e.g. the functionality for gene lists).\nThe conclusions about the tool and its performance are partly supported by the findings presented in the article. Some terms such as \"medF1-score\" and \"accuracy\" are left undefined, and some results omit some methods (Figure 4A has different methods than B or C). Readers may have difficulty understanding the specific questions that were asked and what results are shown.\nMain comments:\nThe clarity of the manuscript can be increased by adding more verbose details about all analyses. Please consider expanding details about each question, the approach, the datasets used, and the results.\n\nPlease consider adding a table describing the reference datasets used in this article, just like the one shown on one of your GitHub repositories. This should help to summarize which datasets were used for the analyses in this article.\n\nComments about specific parts of the manuscript are below. Excerpts from the article are shown in \"quoted italics\" after a bullet point, and my comments are shown directly below the bullet point.\n\n\"A key challenge in scRNA-seq data analysis is the identification of cell types from single-cell transcriptomes. Manual inspection of the expression patterns from a small number of marker genes is still standard practice, which is cumbersome and frequently inaccurate.\"\nDo we know the accuracy by manual inspection? Is there a reference for this? In the absence of evidence, you might consider weakening the statement to say \"may be inaccurate\" rather than \"is cumbersome and frequently inaccurate\". You might consider that many scRNA-seq experiments are done for the purpose of discovering new cell types that have not been well-described in previous published datasets. In this setting, manual inspection is necessary and automated analyses could be inaccurate or misleading.\n\n\"Currently, multiple cell type assignment packages exist but they are specifically tailored towards input types or workflows8–13.\"\nPlease consider naming and describing each method that will be compared to clustifyr in this manuscript, so the reader can assess how the methodology of clustifyr compares to other methods. Which methods are \"specifically tailored towards input types or workflows\"? Could you give an example to help the reader understand this claim?\n\nSuggested improvements for Figure 1:\nIn Figure 1, you might consider showing the dimensions of the inputs and outputs. This might help the reader to understand how they relate to each other.\n\nShould the query and reference data be counts? CPM? Or Log2(CPM + 1)? You might consider elaborating on this.\n\nSuggested improvements for Figure 2:\nPlease consider rotating Figure 2A, D, and E 90 degrees clockwise to improve legibility.\n\nPlease consider limiting the axes ranges to the data instead of using the range [0, 1].\n\nPlease consider increasing all font sizes in all panels in all figures, including titles, legends, axis text, etc. Some readers might need larger sizes to see clearly.\n\nPlease consider changing the title to \"All genes (n = 10,000)\" and \"M3Drop variable genes (n = 1,000)\" in Figure 2C, so we have some sense of the number of genes used to generate each heatmap.\n\nPlease consider showing a graphical representation of the experiment setup for this figure. What is the reference? What is the query? What are their dimensions? What is the main question in this analysis?\n\nOne way to enhance clarity is to add descriptive titles to every figure in every panel (e.g. \"Testing different correlation statistics\", etc.).\n\nPlease consider adding more details to the legend text for Figure 2 to help readers understand exactly what experiment has been done, what data was used, and what result is shown.\n\nIn Figure 2C, it seems that the y-axis and x-axis have been swapped by mistake. I see that the y-axis is labeled \"ground truth cell type\" but it includes \"unclassified\". I would expect the category \"unclassified\" to appear in the \"called cell type\" axis, but not in the \"ground truth cell type\" axis. Are the axes swapped or are they correct? Could you please clarify?\n\nIn Figure 2E, what does the color indicate? Is it the power argument \"n^x\" or something else?\n\nThe reader may be wondering:\nHow many query cells did you use? How many clusters were in the query dataset? How many cells per cluster? How many reference datasets were used? How many clusters were in the reference dataset? Were the query and reference datasets acquired from the same tissue sample or were they completely independent and unrelated?\n\nIn the section \"Subclustering\", please consider adding more details to help the reader avoid misunderstandings. What exactly is the \"sub-clustering power argument (x)\"? Please consider giving a concrete example to help the reader understand this section. Please consider creating a new figure that helps the reader to understand the \"subcluster()\" functionality.\nWhat is the PBMCbench data? Is this the same data as mentioned in the section \"Correlation minimum cutoff\"?\n\nIn the section \"Cells per cluster\", you might consider introducing the dataset, then introducing the question that is being addressed, and finally reporting the results. What is the number (15, 8, 4)? Is the \"Mouse Cell Atlas\" the same as the \"Tabula Muris\"? Were these mouse datasets used in the previous sections? The reader might benefit from an introduction of these datasets.\n\nSuggested improvements for Figure 3:\nPlease consider adding labels \"A\", \"B\", \"C\", \"D\" to mark each of the four panels, so they can be referenced clearly.\n\nPlease consider using the same name consistently in the text and the figure titles. For example, the figure says \"Bulk RNA-seq reference data\" but the text says \"ImmGen database\". The reader might better understand the results if the same label were used in both places instead of using two different labels for the same thing.\n\nPlease consider including the identifiers for readers who wish to find these datasets and download them. For example, if the datasets are available on NCBI GEO, please consider including the accession numbers directly in the legend text, or in a table. Check to see if any other database provides an accession number. If an accession number is not available, please consider providing the DOI for a publication or a URL for a website that provides the data. By the way, if any data you are using is not deposited to a permanent repository, please consider uploading this data to a permanent repository (e.g. Figshare).\n\nIn the section describing Figure 4A, please consider these suggested changes:\nPlease explain what is \"clustifyr\", \"clustifyr_lists\", and \"clustifyr_m3drop\". How was feature selection performed for each analysis in Figure 4A? What is the strategy used by scmap? What is the strategy used by \"Seurat\"? What is the strategy used by \"SingleR\"? How is clustifyr similar or different?\n\nThis section says \"Correlation-based clustifyr classification performed better than hypergeometirc-based gene list enrichment as implemented in clustify_lists.\" Please consider explaining the \"clustify_lists\" algorithm in detail and also consider sharing the quantification of the performance of each approach so the reader can interpret the claim \"performed better\". Also consider elaborating on \"performed better\".\nWhat is \"scRNAseq_Benchmark subsampling\"? Could you elaborate on what this is and why it was used?\n\nSuggested improvements for Figure 4:\nPlease consider including an overview schematic to help the reader understand which datasets were used for each result. Please define \"accuracy\". What is the algorithm for computing this number? Please define \"medF1-score\". What is the algorithm for computing this number? For the lower half of panel B, please consider using a format similar to the one in Figure 2B from Kiselev et al. (20181). For example, please use a log10 axis for time, so readers can see the difference between methods. Why is \"medF1-score\" used for Figure 4C and \"accuracy\" for Figure 4B?\n\nWhy does Figure 4A have 6 methods, Figure 4B have 5 methods, and Figure 4C have 3 methods? Is it possible to include all 6 methods for all panels? Could you please comment on the reasons for excluding or including methods in each analysis?\n\"As we and others observe25, novel algorithms may not be necessary for cell type classification, at least within the current limitations of sequencing technology and our broadstroke understanding of cell “types”. Rather, the generation of community curated reference databases is likely to be critical for reproducible annotation of cell types in scRNA-seq datasets.\"\nI agree that a community curated reference database would be a valuable contribution to the field. You might consider creating a table or other type of descriptive listing that helps the reader to understand all of the references that were used in this article. Consider including tissue source, healthy or disease status, number of cells and genes, technology used for the assay, DOI, data URL, NCBI GEO accession, or any other details that the reader might find helpful.\nThank you for providing a GitHub repository with data files! Please also consider sharing the same data in compressed plain text format (e.g. \"file.tsv.gz\"). In addition to GitHub, please consider using a specialty service that is funded for the purpose of permanently archiving research data such as NIH Figshare (https://nih.figshare.com). There are other options (Zenodo, Open Science Framework OSF, etc.).\n\"As an alternative, clustifyr also supports per-cell annotation, however the runtime is greatly increased and the accuracy of the cell type classifications are decreased due to the sparsity of scRNA-seq datasets, and requires a consensus aggregation step across multiple cells to obtain reliable cell type annotations.\"\nYou might consider offering another alternative option. One extreme is to use the cluster averages, while the other extreme is to use single cells. Perhaps there might be a middle ground where clustifyr could automatically use k-means or some other algorithm to form clusters within the user-defined clusters. This would give the user even more flexibility.\nAfter reviewing the code, I can see that there is an \"overcluster()\" function that seems to do exactly what I suggested. Please consider describing this in the article and showing an example of how it works. In retrospect, I can see that the section titled \"Subclustering\" was supposed to describe this topic — I misunderstood this section on the first read.\nYou may want to double-check all of the links in all of your HTML pages. I see three URLs:\nhttps://github.com/rnabioco/clustifyrdatahub/ https://github.com/rnabioco/clustifyr https://github.com/rnabioco/clustifyrdata\n\nI can see that the \"clustifyrdatahub\" repo has code for creating \".rda\" files from the reference datasets.\nI also see similar scripts at https://github.com/rnabioco/clustifyrdata/tree/master/data-raw\nReaders might be confused when they see two different repos with similar scripts. You might consider deleting the \"clustifyrdatahub\" repo if it is not necessary.\nI'm happy to see that the data is organized and annotated in the GitHub repo. Specifically, in the GitHub \"clustifyrdata\" repo, in the \"README.md\" file, the table shows the name of the reference, the number of cell types, the number of genes, the organism, and a link to the publication. Please consider adding some version of this table to the article, so the reader can understand the scope of this article.\nAfter reviewing the code, I was able to resolve some of my misunderstandings caused by lack of clarity in the terse descriptions in this article. To reduce the chance of misunderstanding by other readers, you might consider clarifying or adding details to the descriptions of functions and results. For example, the article does not mention that GSEA is used to work with gene lists.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "5695", "date": "16 Jul 2020", "name": "Kent Riemondy", "role": "Author Response", "response": "We thank the reviewer for their detailed suggestions which we believe have substantially improved the clarity of the manuscript. Our responses are indicated in italics below. The authors describe an R package for annotating cell clusters in scRNA-seq datasets. Specifically, the package implements code for computing correlations between the columns of two data matrices. They show that high correlations between unknown cell clusters in the first data matrix and annotated cell types in the second matrix can be used to label the unknown cell clusters. They try varying parameters and show the effects on the results, and they also benchmark the time and accuracy compared to other packages designed to annotate scRNA-seq data.Details of the code, methods, and analyses are partly provided. Some details seem to be missing (e.g. the functionality for gene lists).The conclusions about the tool and its performance are partly supported by the findings presented in the article. Some terms such as \"medF1-score\" and \"accuracy\" are left undefined, and some results omit some methods (Figure 4A has different methods than B or C). Readers may have difficulty understanding the specific questions that were asked and what results are shown.Main comments: The clarity of the manuscript can be increased by adding more verbose details about all analyses. Please consider expanding details about each question, the approach, the datasets used, and the results. In an effort to more clearly present clustifyr we have added additional details about each dataset, the questions posed by the analysis, and the conclusions from each analysis. Please consider adding a table describing the reference datasets used in this article, just like the one shown on one of your GitHub repositories. This should help to summarize which datasets were used for the analyses in this article. We have added a table to the main text (Table 1) and a supplemental table that provide additional details about each dataset, and provide a reference of each dataset used in each figure panel.Comments about specific parts of the manuscript are below. Excerpts from the article are shown in \"quoted italics\" after a bullet point, and my comments are shown directly below the bullet point. \"A key challenge in scRNA-seq data analysis is the identification of cell types from single-cell transcriptomes. Manual inspection of the expression patterns from a small number of marker genes is still standard practice, which is cumbersome and frequently inaccurate.\" Do we know the accuracy by manual inspection? Is there a reference for this? In the absence of evidence, you might consider weakening the statement to say \"may be inaccurate\" rather than \"is cumbersome and frequently inaccurate\". You might consider that many scRNA-seq experiments are done for the purpose of discovering new cell types that have not been well-described in previous published datasets. In this setting, manual inspection is necessary and automated analyses could be inaccurate or misleading. To our knowledge there has not been a direct study of the accuracy of manual inspection compared to automated methods. We thank the reviewer for noting this point and have weakened this statement accordingly. We also have noted that automated methods can supplement manual inspection of markers to provide additional justification of the discovery of novel cell types. \"Currently, multiple cell type assignment packages exist but they are specifically tailored towards input types or workflows8–13.\" Please consider naming and describing each method that will be compared to clustifyr in this manuscript, so the reader can assess how the methodology of clustifyr compares to other methods. Which methods are \"specifically tailored towards input types or workflows\"? Could you give an example to help the reader understand this claim?We have added descriptions of the methodologies used by tools that we compared clustifyr against (see Introduction). We also have noted which tools are tailored towards input types: reference single cell data (Seurat, ACTINN, scPred) or workflows: using Seurat objects (Seurat), using singleCellExperiment (singleR, scPred), or using the command-line (ACTINN). Suggested improvements for Figure 1: In Figure 1, you might consider showing the dimensions of the inputs and outputs. This might help the reader to understand how they relate to each other. We have added the dimensions to provide clarity. Should the query and reference data be counts? CPM? Or Log2(CPM + 1)? You might consider elaborating on this. Clustifyr supports both raw counts or log normalized values. The decision of which to use is left to the user and we recommend using similar normalization as used for the reference matrix, if possible. We have added text (under Variable gene selection and normalization) to provide guidance to the user.Suggested improvements for Figure 2: Please consider rotating Figure 2A, D, and E 90 degrees clockwise to improve legibility. We have amended the figures as suggested. Please consider limiting the axes ranges to the data instead of using the range [0, 1]. We respectfully decline to implement this suggestion, as we believe restricting the plot to only the range of the data can over-emphasize minor differences in distributions. Please consider increasing all font sizes in all panels in all figures, including titles, legends, axis text, etc. Some readers might need larger sizes to see clearly. We have increased the font sizes accordingly. Please consider changing the title to \"All genes (n = 10,000)\" and \"M3Drop variable genes (n = 1,000)\" in Figure 2C, so we have some sense of the number of genes used to generate each heatmap. We have changed these titles as suggested. Please consider showing a graphical representation of the experiment setup for this figure. What is the reference? What is the query? What are their dimensions? What is the main question in this analysis? We have added additional details about the query and reference datasets in the main text, legends, and in the titles of figure panels as an alternative to graphical representations. The questions addressed by each analysis are more clearly stated when introducing each figure panel. We believe that these edits now provide sufficient clarity for the reader to understand the content of each figure. One way to enhance clarity is to add descriptive titles to every figure in every panel (e.g. \"Testing different correlation statistics\", etc.). We thank the reviewer for this suggestion and we have added titles to figure panels that we believe were unclearly presented. Please consider adding more details to the legend text for Figure 2 to help readers understand exactly what experiment has been done, what data was used, and what result is shown. We have added additional details about each panel to the legend text as well as additional text to the main manuscript as requested. In Figure 2C, it seems that the y-axis and x-axis have been swapped by mistake. I see that the y-axis is labeled \"ground truth cell type\" but it includes \"unclassified\". I would expect the category \"unclassified\" to appear in the \"called cell type\" axis, but not in the \"ground truth cell type\" axis. Are the axes swapped or are they correct? Could you please clarify? The unclassified cell type was annotated as unclassified in the original study, whereas the query dataset contained a cell type (schwann cells) that appears to be similar to the reference data “unclassified”. We have added additional text to the legend to clarify. In Figure 2E, what does the color indicate? Is it the power argument \"n^x\" or something else? The color did not indicate any particular class and therefore was removed. The reader may be wondering: How many query cells did you use? How many clusters were in the query dataset? How many cells per cluster? How many reference datasets were used? How many clusters were in the reference dataset? Were the query and reference datasets acquired from the same tissue sample or were they completely independent and unrelated? We have added a supplemental table (supplemental table 1) with additional details for each dataset in the manuscript. The tissues samples used for the query and reference datasets were derived from unrelated individuals or mice based on our reading of the original publications for each dataset. An exception was the PBMC-bench dataset, in which multiple single cell technologies were tested using the same aliquot of PBMCs. We have added text to the results section to clarify (under Correlation method). In the section \"Subclustering\", please consider adding more details to help the reader avoid misunderstandings. What exactly is the \"sub-clustering power argument (x)\"? Please consider giving a concrete example to help the reader understand this section. Please consider creating a new figure that helps the reader to understand the \"subcluster()\" functionality.We have added an additional figure (2E) to demonstrate the utility of the subcluster/overcluster_test functionality. What is the PBMCbench data? Is this the same data as mentioned in the section \"Correlation minimum cutoff\"? Yes this is the same dataset. We have added additional details to the text to introduce this dataset, as well as additional details provided in table 1. In the section \"Cells per cluster\", you might consider introducing the dataset, then introducing the question that is being addressed, and finally reporting the results. What is the number (15, 8, 4)? Is the \"Mouse Cell Atlas\" the same as the \"Tabula Muris\"? Were these mouse datasets used in the previous sections? The reader might benefit from an introduction of these datasets.We have provided additional text to the manuscript to introduce and describe these datasets to improve clarity about the analyses conducted. The x axis refers to the number of cells per cluster. Suggested improvements for Figure 3: Please consider adding labels \"A\", \"B\", \"C\", \"D\" to mark each of the four panels, so they can be referenced clearly. We have added these labels and referenced them in the updated figure legend. Please consider using the same name consistently in the text and the figure titles. For example, the figure says \"Bulk RNA-seq reference data\" but the text says \"ImmGen database\". The reader might better understand the results if the same label were used in both places instead of using two different labels for the same thing. We have added subtitles to each panel to more clearly reference the datasets in the text. Please consider including the identifiers for readers who wish to find these datasets and download them. For example, if the datasets are available on NCBI GEO, please consider including the accession numbers directly in the legend text, or in a table. Check to see if any other database provides an accession number. If an accession number is not available, please consider providing the DOI for a publication or a URL for a website that provides the data. By the way, if any data you are using is not deposited to a permanent repository, please consider uploading this data to a permanent repository (e.g. Figshare). The publicly available datasets are referenced in the Data Availability section, with additional details now provided in Table 1. GEO accession numbers, DOIs, or URLs are provided, depending on the datasource. Additionally to further ease access to these resources we have organized these datasets into an ExperimentHub (clustifyrdatahub) that is in the process of being submitted to bioconductor. In the section describing Figure 4A, please consider these suggested changes: Please explain what is \"clustifyr\", \"clustifyr_lists\", and \"clustifyr_m3drop\". How was feature selection performed for each analysis in Figure 4A? What is the strategy used by scmap? What is the strategy used by \"Seurat\"? What is the strategy used by \"SingleR\"? How is clustifyr similar or different? We have added an additional paragraph to explain the differing clustifyr methods shown in Figure 4A. Feature selection was performed by the Tabula muris investigators using the variable genes selected by Seurat by examining a plot of the gene expression mean vs. variance (mean.var.plot). Seurat and clustifyr use these variable genes, whereas SingleR and scmap define variable genes using differential expression testing or M3Drop respectively. We have added text to explain the feature selection methods used by each benchmarked method. This section says \"Correlation-based clustifyr classification performed better than hypergeometirc-based gene list enrichment as implemented in clustify_lists.\" Please consider explaining the \"clustify_lists\" algorithm in detail and also consider sharing the quantification of the performance of each approach so the reader can interpret the claim \"performed better\". Also consider elaborating on \"performed better\".We have elaborated on the clustifyr_lists approach for classifying cell types based on gene set enrichment in the text. Additionally we have included a comparison of two approaches that performed best in our benchmarking, using hypergeometric tests, or using the jaccard index and selecting the cell type with the highest index value (Figure 4A). What is \"scRNAseq_Benchmark subsampling\"? Could you elaborate on what this is and why it was used?We have added additional text to the result section to introduce this dataset. This dataset contains random subsets of the tabula muris dataset to enable investigation of performance and accuracy with varying cell numbers. Suggested improvements for Figure 4: Please consider including an overview schematic to help the reader understand which datasets were used for each result. We have added descriptive titles and additional text to the results section to describe the datasets and goals of each benchmarking test. Please define \"accuracy\". What is the algorithm for computing this number? Accuracy is defined as the ratio between the number of correctly classified clusters and the overall number of clusters for every dataset pair. Please define \"medF1-score\". What is the algorithm for computing this number? medF1-score was a shortened term for median F1-score. We have removed all references to medF1-score and replaced with median F1-score. F1-score, the harmonic mean of the precision and recall, is calculated for each cell type. A median F1-score is reported for every dataset pair. For the lower half of panel B, please consider using a format similar to the one in Figure 2B from Kiselev et al. (20181). For example, please use a log10 axis for time, so readers can see the difference between methods. We have modified the figure accordingly. Why is \"medF1-score\" used for Figure 4C and \"accuracy\" for Figure 4B? A F1-score cannot be calculated when the query and reference datasets contain different cell types. Therefore when comparing datasets with varying cell composition we instead utilized an accuracy metric (as defined above). We have added text to the manuscript that defines accuracy and median F1-score (Benchmarking methods), identifies which datasets were compared with each metric, and provides an explanation of why certain datasets were characterized with accuracy or F1-score. Why does Figure 4A have 6 methods, Figure 4B have 5 methods, and Figure 4C have 3 methods? Is it possible to include all 6 methods for all panels? Could you please comment on the reasons for excluding or including methods in each analysis?We agree that it is confusing for differing tools to be shown in different panels. We have therefore benchmarked these methods in a more consistent fashion to enable comparison of each method across different benchmarking tests. One exception however is scPred, which we were unable to successfully run on the Allen Brain Institute atlas data (Figure 4C), which we have noted in the figure legend. \"As we and others observe25, novel algorithms may not be necessary for cell type classification, at least within the current limitations of sequencing technology and our broadstroke understanding of cell “types”. Rather, the generation of community curated reference databases is likely to be critical for reproducible annotation of cell types in scRNA-seq datasets.\" I agree that a community curated reference database would be a valuable contribution to the field. You might consider creating a table or other type of descriptive listing that helps the reader to understand all of the references that were used in this article. Consider including tissue source, healthy or disease status, number of cells and genes, technology used for the assay, DOI, data URL, NCBI GEO accession, or any other details that the reader might find helpful.In addition to the dataset details provided in the Data Availability section and the details provided in Table 1, we have also included a supplemental table that references the datasets used in each figure, and an ExperimentHub package allowing direct access to these resources in R. Thank you for providing a GitHub repository with data files! Please also consider sharing the same data in compressed plain text format (e.g. \"file.tsv.gz\"). In addition to GitHub, please consider using a specialty service that is funded for the purpose of permanently archiving research data such as NIH Figshare (https://nih.figshare.com). There are other options (Zenodo, Open Science Framework OSF, etc.).The datasets used in this study were all published by other research groups and are hosted in various data repositories including GEO and figshare. As mentioned above we have provided additional methods to access these published and publicly available resources. \"As an alternative, clustifyr also supports per-cell annotation, however the runtime is greatly increased and the accuracy of the cell type classifications are decreased due to the sparsity of scRNA-seq datasets, and requires a consensus aggregation step across multiple cells to obtain reliable cell type annotations.\" You might consider offering another alternative option. One extreme is to use the cluster averages, while the other extreme is to use single cells. Perhaps there might be a middle ground where clustifyr could automatically use k-means or some other algorithm to form clusters within the user-defined clusters. This would give the user even more flexibility.After reviewing the code, I can see that there is an \"overcluster()\" function that seems to do exactly what I suggested. Please consider describing this in the article and showing an example of how it works. In retrospect, I can see that the section titled \"Subclustering\" was supposed to describe this topic — I misunderstood this section on the first read.We have added an additional figure panel (Figure 2E) to illustrate this functionality. You may want to double-check all of the links in all of your HTML pages. I see three URLs: https://github.com/rnabioco/clustifyrdatahub/ https://github.com/rnabioco/clustifyr https://github.com/rnabioco/clustifyrdata I can see that the \"clustifyrdatahub\" repo has code for creating \".rda\" files from the reference datasets.I also see similar scripts at https://github.com/rnabioco/clustifyrdata/tree/master/data-rawReaders might be confused when they see two different repos with similar scripts. You might consider deleting the \"clustifyrdatahub\" repo if it is not necessary.We apologize to the reviewer for the confusion of multiple data repositories. We have organized clustifyrdata into an ExperimentalHub Bioconductor package at the request of reviewer #1, resulting in overlapping content in the clustifyrdatahub repository. We have mentioned these differences in the documentation of these repositories and added text to the manuscript to point readers to the experimentHub package, which is currently being submitted to bioconductor. I'm happy to see that the data is organized and annotated in the GitHub repo. Specifically, in the GitHub \"clustifyrdata\" repo, in the \"README.md\" file, the table shows the name of the reference, the number of cell types, the number of genes, the organism, and a link to the publication. Please consider adding some version of this table to the article, so the reader can understand the scope of this article.We have added a table (Table 1) to the main manuscript that contains additional details about each dataset. After reviewing the code, I was able to resolve some of my misunderstandings caused by lack of clarity in the terse descriptions in this article. To reduce the chance of misunderstanding by other readers, you might consider clarifying or adding details to the descriptions of functions and results. For example, the article does not mention that GSEA is used to work with gene lists.We have added additional details about the gene list methods (including GSEA) to the article. clustify() and clustify_lists() are the most important functions implemented in clustifyr, which we believe are now sufficiently described in the revised manuscript. Additional package and function level documentation is provided at https://rnabioco.github.io/clustifyr/ , which we’ve now provided as a link in the software availability section." } ] } ]	1	https://f1000research.com/articles/9-223
https://f1000research.com/articles/9-711/v1	16 Jul 20	{ "type": "Systematic Review", "title": "Early and frequent exposure to antibiotics in children and the risk of obesity: systematic review and meta-analysis of observational studies", "authors": [ "Archita Srivastava", "Kim Chau", "Henry Kwon", "Qin Guo", "Bradley C. Johnston", "Archita Srivastava", "Kim Chau", "Henry Kwon", "Qin Guo" ], "abstract": "Background: This study aimed to systematically evaluate the available evidence on prenatal and early infancy antibiotic exposure and the association with overweight and obesity in later childhood. Methods: We conducted a comprehensive search of Embase, MEDLINE, and Web of Science for observational studies assessing prenatal and early antibiotic exposure on the risk of overweight and obesity. We independently assessed the risk of bias using the ROBINS instrument and the overall quality of evidence using the GRADE approach. Results: Our search identified thirteen observational studies including 554,983 participants; most studies were at moderate risk of bias. We found a statistically significant impact of early antibiotic exposure and the risk of being overweight later in childhood (OR 1.18; 95% CI 1.05 to 1.34) (very low quality evidence). We also found that early childhood antibiotic exposure was associated with the risk for childhood obesity (OR 1.14; 95% CI 1.04 to 1.24) (very low quality evidence). Conclusions: Very low quality evidence suggests that exposure to antibiotics early in life may be associated with an increased risk of being overweight and obese in later childhood. However, very low quality evidence raises serious questions about the plausibility of prenatal and early infancy antibiotic exposure being causally related to weight in children. PROSPERO registration: CRD42016050011 (14/12/2016)", "keywords": [ "Antibiotics", "Early Life", "Obesity", "Overweight", "Prenatal" ], "content": "Introduction\n\nInfants and children are commonly and frequently prescribed antibiotics and up to 40% of infants are exposed either directly or through maternal intra-partum antibiotic prophylaxis1,2. In the United States it is estimated that a child receives approximately three courses of antibiotics by the age of two and 10 courses by the age of 10 years3 and, often, these courses are prescribed for viral infections thus offering no therapeutic benefit4. The concerns with significant overuse of antibiotics are increased antibiotic resistance, increased rates of adverse drug reactions, such as rashes, fungal infections, and antibiotic-associated diarrhea. The intestinal microbiota begins development in utero and resembles adult microbiota by 2.5 years of age4. Thus, external exposures to antibiotics during this phase of microbiota development may potentially impact the normal colonization pattern, and the composition of the gut microbiota, both of which may play a role in health outcomes later in life, including weight gain and obesity2,3,5.\n\nWhile the paradigm has been that the infant gut is sterile at birth, increasing evidence suggests that colonization may begin in utero with bacterial colonies detected in the placenta and meconium6. Thus, it is possible that prenatal or intrapartum antibiotic exposure may potentially affect these bacterial colonies in utero. Most notably, antibiotic exposure can affect the gut microbiota composition at any age; however, infants may especially be vulnerable, as the gut microbiota is highly unstable and dynamic during this period with greater interindividual variability, compared to that of an adult7–9. Cho et al. and Cox et al. have shown increased fat accumulation in mice who were treated with antibiotics early in age regardless of antibiotic class10–12. This corresponds with an absence of certain populations of prominent microbes, such as Lactobacillus, Allobaculum, Rikenellaceae, and Candidatus Arthromitus due to antibiotic exposure. This suggests a potential protective role of these bacteria against patient-important outcomes, such as weight gain and obesity11,12. As such, this paper aims to evaluate and summarize the available evidence on the potential impact of prenatal, intrapartum, and early childhood antibiotic exposure on the risk of overweight and obesity later in life.\n\n\nMethods\n\nThis review was registered with PROSPERO - International Prospective Register of Systematic Reviews on the 14th December 2016 under the number CRD4201605001113.\n\nAn experienced clinical librarian (TAW) conducted a literature search in the following electronic databases from inception to June 2018: Embase (Ovid), MEDLINE (Ovid), MEDLINE In-Process & Other Non-Indexed Citations (Ovid), MEDLINE® Epub Ahead of Print (Ovid), and Web of Science. The search terms included database-controlled vocabulary and keywords for the concepts of “antibacterial agents” AND “children” AND “microbiome” OR “microbiota” AND “health outcomes” (e.g. weight, obesity, diabetes). We also supplemented the search by reviewing bibliographies of review articles and other eligible clinical studies to ensure that studies that were not identified by the search strategy were included. Clinicaltrials.gov was searched for unpublished and ongoing trials. No language or date restrictions were applied. See extended data for full search strategies14.\n\nInclusion criteria. Observational and experimental study designs were eligible, including cohort, cross-sectional, and case-control studies, data modelling studies, and randomized controlled trials. Further, to be eligible, studies had to examine maternal prenatal, intrapartum, and child (birth to 18 years) exposure to antibiotics and at least one of our target health outcomes of interest: overweight (body mass index [BMI] ≥ 25 kg/m2), or obesity (BMI ≥ 30 kg/m2). The comparison group included children that were not exposed to antibiotics. Studies with both fixed and variable follow-up periods were eligible. Studies or subgroups within studies that included premature infants, infants born with low birth weight or comorbidities, such as cystic fibrosis and Crohn’s disease were excluded.\n\nScreening, data extraction and quality assessment. Blinded to the journal of publication and results, two teams of independent reviewers (AS with either LL or HK) screened titles and abstracts of the studies to determine eligibility. Full-text articles were retrieved and assessed for further eligibility assessment. Discrepancies were resolved by discussion and, when necessary, additional input from a third reviewer (BCJ, KC).\n\nTwo reviewers (AS with either KC or HK) extracted data independently using a standardized data extraction form. The following data was extracted: study design, study setting, demographic information, antibiotic regimen (frequency and duration of exposures, type of antibiotic/class), and outcome data for each of our target outcomes including reported time points and duration of follow-up.\n\nAs no randomized trials were identified, the Risk of Bias In Non-randomized Studies (ROBINS) instrument for different types of observational studies15 was used to assess study validity. Two reviewers (AS and KC) independently evaluated each observational study included for risk of bias (RoB). The ROBINS instrument consists of seven total RoB questions, with two questions under the pre-intervention domain, one question on the intervention domain and four questions under the post-intervention domain15. Response options for each question included ‘low RoB’, ‘moderate RoB’, ‘serious RoB’, and ‘critical RoB’. We modified the instrument; wherein low and moderate risk of bias was classified as ‘low risk of bias’ and serious and critical risk of bias was classified as ‘high risk of bias’. For each study, if one or more questions was judged to be high or critical risk of bias, the overall study was deemed to be at high risk of bias15. Any disagreements regarding data extraction or risk of bias items was resolved through discussion, and, when necessary, with an experienced methodologist (BCJ).\n\nWe applied the GRADE (Grading of Recommendations, Assessment, Development, and Evaluations) approach16 for rating the overall quality of evidence for the outcomes of interest. In particular, observational studies were considered low quality evidence, but may be rated up for three reasons: (1) when a large magnitude of effects exists (e.g. OR <0.5 or >2.0), (2) when there is a dose-response gradient or (3) when all plausible confounding or other biases may be working against the observed effect16. Observational studies without these characteristics were considered low quality evidence. In addition, if studies were limited by risk of bias, inconsistency, indirectness, imprecision or publication bias the overall quality of evidence may to rated down to very low quality17. The quality of evidence for each main outcome was determined after considering each of these elements, and categorized as either high (highly confident that the true effect lies close to that of the estimate of the effect); moderate (moderately confident in the effect estimate: the true effect is likely to be close to the estimate of the effect, but there is a possibility that it is substantially different); low (confidence in the effect estimate is limited: the true effect may be substantially different from the estimate of the effect); very low (very little confidence in the effect estimate: the true effect is like to be substantially different from the estimate of effect)17.\n\nMeta-analysis was conducted using Stata 14 (Stata Corp., College Station, TX, USA). Effect estimates and corresponding confidence intervals were extracted from eligible articles and we calculated the adjusted odds ratio (OR) with corresponding 95% confidence interval with the generic inverse variance method was used to determine relative effects18.\n\nWe conducted two primary meta-analyses, one for overweight and one for obesity with obesity including overweight. Random effects models were used due to the anticipated heterogeneity between studies. We used the DerSimonian and Laird method for estimating tau-squared and subsequent adjustment for effect size19.\n\nHeterogeneity was assessed with the I-squared measure and the corresponding Q statistical test18. To further explore heterogeneity, we conducted a priori subgroup and sensitivity analyses20. Our three subgroups of interest included (1) the timing of exposure (prenatal versus early exposure), (2) number of antibiotic exposures (1 to 2 versus 3 or more), and (3) time point of outcome assessment (less than 7 years versus 7 years of age or more). Subgroups on timing and number of antibiotic exposures were stated a priori13, while the time point of assessment was a post-hoc subgroup chosen based on the distribution of outcome assessments among eligible studies. We also conducted a sensitivity analysis on study design limitations (risk of bias). Low and moderate risk of bias was classified as low risk of bias and serious and critical risk of bias was classified as high risk of bias.\n\n\nResults\n\nAmong 12,091 articles identified, 13 studies were deemed eligible for this review (Figure 1), including 11 cohort studies2,21–25,26–30, one cross-sectional study31, and one nested case-control study1. Five studies analyzed the effect of prenatal exposure to antibiotics23,24,26,30,31 while the remaining eight studies evaluated the impact of early childhood (birth to 2 years) antibiotic exposure and the risk of overweight and obesity1,2,21,22,25,27–29. Included studies ranged in size from 9729 to 312,702 participants25 with a total sample size among all included studies of 554,983. The median age for weight assessment was 7 years (range 2 years to 18 years). A detailed summary of all 13 study characteristics is shown in Table 1.\n\nSES = socioeconomic status; BMI = body mass index\n\nUsing ROBINS instrument to independently assess risk of bias for weight-related outcomes, 11 studies were rated as ‘moderate’ for risk of bias1,21–25,27–30,31 and two studies were rated as ‘serious’ for risk of bias2,26. The studies rated as serious risk of bias were missing significant participant outcome data, with exclusion of participants introducing bias in the study. Despite all studies having adjusted for potential confounders, each included study was rated as moderate risk of bias for confounding. The choice and number of variables adjusted for differed considerably between studies. In some instances, the adjustment factors were theoretically not potential confounders (e.g. ethnicity), while in other instances, potential confounders such as socioeconomic status were not adjusted for. The complete risk of bias assessment for each individual study is provided in Table 2.\n\na Confounding: One or more prognostic variables also predicts the intervention received at baseline.\n\nb Selection Bias: When exclusion of some eligible participants is related to both intervention and outcome, there will be an association between interventions and outcome even if the effect of interest is truly null.\n\nc Information Bias: Bias introduced by either differential or non-differential misclassification of intervention status.\n\nd Confounding: Bias that arises when there are systematic differences between experimental intervention and comparator groups in the care provided, which represent a deviation from the intended intervention.\n\ne Selection Bias: Bias that arises when later follow-up is missing for individuals initially included and followed (e.g. differential loss to follow-up that is affected by prognostic factors)\n\nf Information Bias: Bias introduced by either differential or non-differential errors in measurement of outcome data. Such bias can arise when outcome assessors are aware of intervention status, if different methods are used to assess outcomes in different intervention groups, or if measurement errors are related to intervention status or effects.\n\ng Reporting Bias: Selective reporting of results from among multiple measurements of the outcome, analyses or subgroups in a way that depends on the findings.\n\nWe found statistically significant effect of antibiotics on the risk of being overweight (OR 1.18; 95% CI 1.05 to 1.34, 8 studies, 125,533 participants, I2=58.5%) (Figure 2). Using the estimated population risk of being overweight32, the risk difference between exposed and non-exposed was equivalent to 26 more overweight cases per 1000 children (95%CI 7 more to 47 more cases). Our GRADE assessment indicates that the overall quality of evidence for the risk of being overweight was very low due to serious indirectness (Table 3).\n\n1 This control group estimate is the risk of being overweight or obese in children 5 to 19 years old. The risk estimate comes from the WHO website (https://www.who.int/news-room/fact-sheets/detail/obesity-and-overweight).\n\n2 Seven studies were judged to be moderate risk of bias, while one was judged to be serious risk of bias and we did not downgrade for study limitations\n\n3 Heterogeneity among eight studies (P = 0.018, I2 = 58.5%) was considered to be moderate according to the Cochrane Handbook (2011). All studies however had over lapping 95%CIs and demonstrated the same direction of effect. We therefor chose to not downgrade.\n\n4 With respect to indirectness issues, participants exposed were different, wherein 4 studies included infants (post-natal), while the other 4 studies included mothers (pre-natal) as the population exposed to antibiotics. In our subgroup assessment of post-natal versus pre-natal, we found no significant difference between groups indicating limited evidence to suggest that timing of exposure impacts weight (P = 0.518). With respect to timing of assessment, there were different time points among 8 studies, ranging from 1 year age to 16 years of age. Our subgroup analysis on the timing of assessment, although dichotomized due to lack of power (<7 years versus ≥ 7 years), showed no significant difference (P = 0.699). With respect to measuring weight, there were 5 different definitions (e.g. BMI from 25 to <30; BMI > 85 percentile) of documenting weight among 8 studies. We decided to downgrade for serious indirectness related to the measurement of weight, particularly because we could not conduct an appropriate subgroup analysis (i.e. conduct meta-regression or dichotomize).\n\n5 The results are precise and we did not downgrade for imprecision.\n\n6 Given there were fewer than 10 studies we could not assess for publication bias.\n\n7 With respect to the size of the effect related to antibiotic exposure, all studies consistently demonstrating an odds ratio of < 2 and we did not upgrade.\n\n8 In our subgroup assessment of those receiving 1 to 2 antibiotic exposures versus those receiving 3 or more, we found no evidence of an increased risk with an increased dose and we therefor did not rate up for dose response.\n\n9The risk estimates come from WHO website (https://www.who.int/news-room/fact-sheets/detail/obesity-and-overweight) and the Non-Communicable Diseases (NCD) Risk Factor Collaboration (Lancet 2017 Dec 16;390(10113):2627-2642).\n\n10 Six of eight studies were judged to be at moderate risk of bias overall risk of bias and we did not downgrade for study limitations.\n\n11 Substantial heterogeneity among studies (P < 0.001, I2 = 76.8%). Although all studies have same direction of effect, the 95%CIs do not fully overlap. Further, our subgroup analysis did not explain the observed heterogeneity and hence we downgraded.\n\n12 With respect to indirectness issue, participants exposed were different, wherein 5 studies included infants (post-natal), while the other 3 studies included mothers (pre-natal) as the population exposed to antibiotics. In our subgroup assessment of post-natal versus pre-natal, we found no significant difference between groups indicating limited evidence to suggest that timing of exposure impacts weight (P = 0.353). With respect to timing of assessment, there were different time points among 8 studies, ranging from 2 years age to 16 years of age. Our subgroup analysis on the timing of assessment, although dichotomized due to lack of power (< 7 years versus ≥ 7 years), showed no significant difference (P = 0.853). With respect to measuring the potential impact of antibiotics on weight, there were 3 different definitions (e.g. BMI ≥30; BMI > 95 percentile) of documenting weight among 8 studies and the reference control group was both normal weight, normal weight plus overweight, and we rated down for indirectness issues.\n\nWe found an overall significant effect of antibiotics on the risk of obesity (OR 1.14; 95% CI 1.04 to 1.24, 7 studies, 497,209 participants, I2=76.8%) (Figure 3). Based on the estimated population risk of obesity in children14, the risk difference between those exposed and non-exposed to antibiotics was equivalent to 9 more obesity cases per 1000 (95%CI 3 more to 15 more cases). Again, our GRADE assessment indicated that the overall quality of evidence for the obesity was very low due to serious inconsistency between studies (Table 3).\n\nPrenatal vs. early infancy antibiotic exposure. For prenatal timing of exposure, the effect of antibiotics on the risk of being overweight was not statistically significant, random effects (OR = 1.13, 95% CI 0.97 to 1.32, 4 studies, 54865 participants, I2=57.5%) (Figure 4). For early infancy exposure, we found a significant effect of antibiotics on the risk of being overweight (OR = 1.26, 95% CI 1.0 to 1.57, 4 studies, 70668 participants, I2=54.8%). Our meta-regression analysis showed no significant difference between subgroups with regard to the effect of antibiotics on the risk of being overweight outcome (p =0.518).\n\nOutcome assessment based on follow-up time point. When the risk was assessed among those less than 7 years, we found a statistically significant effect of antibiotics on this risk of being overweight (OR = 1.22, 95% CI 1.05 to 1.42, 4 studies, 30952 participants, I2=0%). When the risk was assessed at 7 years or more, the effect of antibiotics on the risk of being overweight was not statistically significant, random effects OR = 1.18, 95% CI 0.98 to 1.43, 4 studies, 94581participants, I2=78.1%) (Figure 5). Meta-regression analysis showed no significant difference between subgroups with regards to the effect of antibiotics on the risk of being overweight (p = 0.699).\n\nNumber of antibiotic exposures. For 1 to 2 antibiotic exposures, the effect of antibiotics of antimicrobials on the risk of being overweight was not statistically significant (OR = 1.30, 95% CI 0.94 to 1.81, 3 studies, 53941 participants, I2=84.1%) (Figure 6). For 3 or more antibiotic exposures, the effect of antimicrobials on the risk of being overweight was not statistically significant, random effects (OR = 1.29, 95% CI 0.92 to 1.83, 3 studies, 53941participants, I2=67.7%). Similarly, our meta-regression analysis showed no significant difference between subgroups for antibiotic exposure and overweight risk (p =0.937).\n\nSensitivity analysis: Risk of bias assessment. For low risk of bias studies, we found a statistically significant effect of antibiotics on the risk of being overweight (OR = 1.18, 95% CI 1.02 to 1.37, 7 studies, 110992 participants, I2=61.6%) (Figure 7).\n\nPrenatal vs. early antibiotic exposure. For prenatal timing of exposure, we found a significant effect of antibiotics on obesity (OR 1.32; 95% CI 1.01 to 1.73, 3 studies, 53,945 participants, I2=80.2%) and a non-significant effect for early infancy exposure (OR 1.07; 95% CI 0.99 to 1.16, 5 studies, 443,264 participants, I2=68.4%) (Figure 8). However, subgroup analysis showed no significant difference between prenatal and early infancy subgroups on the risk of obesity (p = 0.353).\n\nTimepoint of outcome assessment. For the time point of outcome assessment (less than 7 years), the effect of antibiotics on obesity risk was not statistically significant (OR 1.14, 95% CI 0.97 to 1.35, 4 studies, 130,562 participants, I2=68.9%), while for 7 years or more, we found a significant effect (OR 1.18, 95% CI 1.01 to 1.38, 4 studies, 366,647 participants, I2=84.1%) (Figure 9). Subgroup analysis showed no significant difference between subgroups (p = 0.853).\n\nNumber of antibiotic exposures. For those with 1 to 2 antibiotic exposures, the risk of obesity was not statistically significant (OR 1.03, 95% CI 0.98 to 1.09, 4 studies, 70,199 participants, I2=0%), while for those with 3 or more exposures, we found no statistically significant effect (OR 1.29, 95% CI 0.92 to 1.83, 4 studies, 70,199 participants, I2= 76.1%) (Figure 10). Again, subgroup analysis showed no significant difference between 1 to 2 exposures and 3 or more exposures (p = 0.085). The subgroup results, however, reached statistical significance with 3 or more antibiotics having a larger associated OR for obesity later in life.\n\nSensitivity analysis: Risk of Bias assessment. For low (6 studies, 48,1941 participants) versus high risk of bias (2 studies, 15268 participants) studies, we found no significant difference between subgroups (p = 0.118) (Figure 11).\n\n\nDiscussion\n\nIn total, 13 observational studies with over 554,983 participants were identified that examined the association between prenatal or early childhood antibiotic exposure and the risk of weight-related outcomes. Overall, based on very low quality evidence, antibiotic exposure may be associated with overweight and obesity. The absolute risk increase between those exposed and non-exposed to antibiotics was equivalent to 26 more overweight cases per 1,000 children followed, and 9 more obesity cases per 1,000 children followed.\n\nWe specified a priori subgroup hypotheses to assess the stability of association across population and intervention variables thought to potentially modify our outcomes of interest. We found a statistically significant association between prenatal antibiotic exposure and obesity, between late antibiotic exposure (>7 years) and obesity and between higher frequency of exposures to antibiotics (3 or more scripts) and the risk of obesity. When we conducted a test of interaction for each of our subgroups, although none were statistically significant, the frequency of exposure was almost significant (p = 0.085), suggesting that higher antibiotic frequency may increase the risk of obesity later in life. This finding corresponds to our a priori hypothesis13 that with higher exposure comes a higher risk of obesity, potentially attributable to a more frequent disruption of the composition of the gut microbiota7–9.\n\nThe quality of evidence for each outcome was determined using the GRADE criteria17. For the primary outcome, overweight, the quality of evidence was categorized as very low because of serious indirectness related to the measurement of weight, there were 5 different definitions (e.g. BMI from 25 to <30; BMI > 85 percentile). For the obesity health outcome, the quality of evidence was categorized as very low due to indirectness (the reference control group was both normal weight and normal weight plus overweight) and inconsistency between studies (P < 0.001, I2 = 76.8%), and our subgroup analyses did not explain the observed heterogeneity. Despite authors’ adjustment for a variety of known or suspected confounders, observational studies are prone to residual confounding and after considering each of the GRADE criteria, including the possibility of rating up for magnitude of effect and for dose-response, the evidence provides only very low quality evidence.\n\nThe association between antibiotic exposure in early life and increased risk of being overweight or obese is also supported by observations from animal studies. A study conducted by Angelakis et al. (2013)33 reported an increase in fat accumulation in mice when the mice were exposed to antibiotics at the weaning stage. The underlying mechanisms may be due to an upregulation of genes involved in lipogenesis, as well as antibiotic-induced changes in gut microbiota composition resulting in increased production of short-chain fatty acid, an additional energy source which results in imbalance of energy regulation, contributing to obesity34,35. In other studies, it has been suggested that microbial colonization may begin in utero through exchange of placental bacteria from mother to fetus; thus, placental transfer of antibiotics consumed during pregnancy may enter fetal circulation resulting in microbial changes similar to that of early life antibiotic exposure10,36.\n\nOur paper has several strengths. First, our review included a systematic literature search of three primary databases and gray literature sources and is the most comprehensive review to date including 13 studies on weight-related outcomes. Second, an a priori design was published as a written protocol and registered with PROSPERO13. We closely followed our a priori protocol, however, we did make some post-hoc analysis and presentation decisions including the addition of a subgroup on follow-up time (up to 6 years vs 7 or more years of age). Third, we independently assessed the quality of the evidence for each outcome using the GRADE approach, allowing us to document the uncertainty we have in attributing antibiotic exposure as a causal risk factor. Fourth, we quantitatively analyzed our a priori subgroups of interest including prenatal versus early infancy antibiotic exposure, and frequency of exposure to explore hypothesized effect modifiers1,22,26, suggesting based on the available data, that frequency of exposure deserves further study.\n\nThis systematic review also has two major limitations. First, the search for included articles in this review was completed in June 2018. However, the discussion (below) has been updated to include recent evidence from studies published since spring 2018. Second, there was significant heterogeneity across the included studies with respect to the number and types of antibiotics used, the timeframe between antibiotic exposure and weight assessment, and the method of weight assessment. Although we minimized confounding by using the most adjusted analyses from each study in our meta-analyses, spurious results due to residual confounding remains a plausible explanation for all associations. That is, given that all the included studies were observational in nature, there is a risk of uncontrolled confounding factors despite multivariable adjustment37.\n\nThe potential relation between early infancy antibiotic exposure and weight issues for children is a burgeoning field of investigation, with new SRMAs and a new cross-sectional study including twins published between 2018–2020. Below, we summarized the consistency and methodological aspects of recent studies including the 2 highest quality SRMAs38,39. In a SRMA conducted by Rasmussen et al. (2018)38, 13 studies were included with 8 studies included in the meta-analysis. The reasons for excluding 5 studies from the quantitative synthesis were: high risk of bias and incomplete data for use in the meta-analysis. The outcomes of interest were childhood weight, obesity and body mass index (BMI). The study concluded that antibiotic exposure in early infancy is associated with slightly higher risk of combined overweight or obesity in childhood (OR 1.11, 95% CI 1.02 to 1.20). The study also conducted a subgroup analysis involving number of exposures to antibiotics and time point of exposure. More than 1 antibiotic treatment among participants was associated with an OR of 1.24 (95% CI 1.09 to 1.43), and exposure within the first 6 months of life was associated with an OR of 1.20 (95% CI 1.04 to1.37). Similarly, Aghaali et al. (2019)39 conducted a systematic review and meta-analysis of 19 total studies. The outcomes of interest were childhood overweight or obesity, difference in childhood body mass index (BMI) and weight between the exposed group and the non-exposed group. The study found a significant association between early childhood antibiotic exposure (<2 years) and risk of childhood weight gain and obesity (OR 1.05, 95% CI 1.04 to 1.06). The study also conducted a subgroup analysis involving time point of exposure. Among infants exposed to antibiotics before 6 months of age, the odds of more weight gain was 11%, while for infants exposed to antibiotics after 6 months of age, the odds of more weight gain was 7%. However, unlike our review, Rasmussen et al. (2018)37 and Aghaali et al. (2019)39 did not conduct a test of interaction for their subgroups based on the number of exposures and timing of exposure, nor was there a study protocol available. In addition to having a publicly available protocol, our study is the only systematic review and meta-analysis to rate the quality of the evidence for each outcome using the GRADE approach, and to present data as an absolute risk difference, an approach to presenting results that has been shown to be more intuitive and helpful for decision-makers40,41.\n\nOur findings must be interpreted in the light of a recent cross-sectional study42 of 284,211 participants that included siblings and twins in New Zealand and that analyzed prenatal and early infancy antibiotic exposure (first two years) and its association with obesity at 4 years. The study found that both prenatal and early infancy exposure to antibiotics were independently associated with obesity at 4 years in a dose dependent manner. For the child’s exposure, the OR for the association between antibiotic exposure and obesity was 1.04 (95%CI 1.03 to 1.05) among siblings and 1.05 (95%CI 1.02 to 1.09) among twins. However, fixed effect analysis of siblings (6249) and twins (522) with discordant outcomes showed no association between antibiotic exposure and obesity, with ORs of 0.95 (95%CI 0.90 to 1.00) for maternal exposure, 1.02 (95%CI 0.99 to 1.04) for early infancy exposure among all children, and 0.91 (95%CI 0.81 to 1.02) for twins’ exposure. The findings by Leong et al. (2020) indicates unmeasured confounding factors in previous studies, and supports our findings, indicating very low quality evidence for a trivial to very small increased risk of weight issues later in life.\n\nOverall, our systematic review suggests that prenatal and early childhood antibiotic exposure is associated with an increased associated risk of overweight and obesity among children and adolescents, potentially independent of more established early determinants of obesity. Our findings suggest that the first few years of life may represent a critical window of development, where external exposures, such as antibiotics, may program metabolic pathways and obesity risk through mechanisms involving the gut microbiota; however, experimental studies are needed to establish the impact of antibiotics, particularly frequent antibiotic use, on the gut microbiota and how this may impact weight-gain in humans. Until registered protocol-driven higher quality cohort studies with explicit plans for adjustment and statistical analysis that better demonstrate an antibiotic dose-response curve or controlled clinical trials (e.g. watchful waiting for otitis media) with long-term follow-up are conducted to confirm or refute these findings, very low quality evidence raises serious questions about the plausibility of prenatal and early antibiotic exposure being causally related to weight in children.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nHarvard Dataverse: Antibiotics and Weight Outcomes, Diabetes and Microbiome Literature Search. https://doi.org/10.7910/DVN/BOGPJL14\n\nThis project contains the following extended data:\n\n- antibiotics_health_outcomes_search_strategy.docx (Search Strategy)\n\nHarvard Dataverse: PRISMA checklist for ‘Early and frequent exposure to antibiotics in children and the risk of obesity: systematic review and meta-analysis of observational studies’ https://doi.org/10.7910/DVN/BOGPJL14", "appendix": "Acknowledgements\n\nWe would like to thank Lyubov Lytvyn for assisting with protocol development and screening of titles and abstracts.\n\n\nReferences\n\nAzad MB, Bridgman SL, Becker AB, et al.: Infant antibiotic exposure and the development of childhood overweight and central adiposity. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHiggins JPT, Green S (editors): Cochrane Handbook for Systematic Reviews of Interventions. Version 5.1.0 [updated March 2011]. The Cochrane Collaboration, 2011. Reference Source\n\nDerSimonian R, Laird N: Meta-analysis in clinical trials. Control Clin Trials. 1986; 7(3): 177–188. PubMed Abstract \| Publisher Full Text\n\nSun X, Briel M, Busse JW, et al.: Subgroup Analysis of Trials Is Rarely Easy (SATIRE): a study protocol for a systematic review to characterize the analysis, reporting, and claim of subgroup effects in randomized trials. Trials. 2009; 10: 101. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAjslev T, Andersen C, Gamborg M, et al.: Childhood overweight after establishment of the gut microbiota: the role of delivery mode, pre-pregnancy weight and early administration of antibiotics. Int J Obes (Lond). 2011; 35(4): 522–529. PubMed Abstract \| Publisher Full Text\n\nBailey L, Forrest C, Zhang P, et al.: Association of Antibiotics in Infancy With Early Childhood Obesity. JAMA Pediatr. 2014; 168(11): 1063–9. PubMed Abstract \| Publisher Full Text\n\nBridgman SL, Azad MB, Persaud RR, et al.: Impact of maternal pre-pregnancy overweight on infant overweight at 1 year of age: associations and sex-specific differences. Pediatric Obesity. 2018; 13(10): 579–89. PubMed Abstract \| Publisher Full Text\n\nCassidy-Bushrow AE, Burmeister C, Havstad S, et al.: Prenatal antimicrobial use and early-childhood body mass index. Int J Obes (Lond). 2018; 42(1): 1–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi DK, Chen H, Ferber J, et al.: Infection and antibiotic use in infancy and risk of childhood obesity: a longitudinal birth cohort study. Lancet Diabetes Endocrinol. 2017; 5(1): 18–25. PubMed Abstract \| Publisher Full Text\n\nMueller NT, Whyatt R, Hoepner L, et al.: Prenatal exposure to antibiotics, cesarean section and risk of childhood obesity. Int J Obes(Lond). 2014; 39(4): 665–670. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaari A, Virta LJ, Sankilampi U, et al.: Antibiotic Exposure in Infancy and Risk of Being Overweight in the First 24 Months of Life. Pediatrics. 2015; 135(4): 617–626. PubMed Abstract \| Publisher Full Text\n\nScott FI, Horton DB, Mamtani R, et al.: Administration of Antibiotics to Children Before Age 2 Years Increases Risk for Childhood Obesity. Gastroenterology. 2016; 151(1): 120–129.e5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVille AP, Heyman MB, Medrano R, et al.: Early antibiotic exposure and risk of childhood obesity in Latinos. Child Obes. 2017; 13(3): 231–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang B, Liu J, Zhang Y, et al.: Prenatal Exposure to Antibiotics and Risk of Childhood Obesity in a Multicenter Cohort Study. Am J Epidemiol. 2018; 187(10): 2159–67. PubMed Abstract \| Publisher Full Text\n\nMor A, Antonsen S, Kahlert J, et al.: Prenatal exposure to systemic antibacterials and overweight and obesity in Danish schoolchildren: a prevalence study. Int J Obes (Lond). 2015; 39(10): 1450–1455. PubMed Abstract \| Publisher Full Text\n\nObesity and overweight. Who.int. 2019; [cited 9 August 2019]. Reference Source\n\nAngelakis E, Merhej V, Raoult D: Related actions of probiotics and antibiotics on gut microbiota and weight modification. Lancet Infect Dis. 2013; 13(10): 889–99. PubMed Abstract \| Publisher Full Text\n\nTurta O, Rautava S: Antibiotics, obesity and the link to microbes-what are we doing to our children? BMC Med. 2016; 14(1): 57. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMurugesan S, Nirmalkar K, Hoyo-Vadillo C, et al.: Gut microbiome production of short-chain fatty acids and obesity in children. Eur J Clin Microbiol Infect Dis. 2017; 37(1): 1–5. PubMed Abstract \| Publisher Full Text\n\nPacifici G: Placental transfer of antibiotics administered to the mother: a review. Int J Clin Pharmacol Ther. 2006; 44(2): 57–63. PubMed Abstract \| Publisher Full Text\n\nTilaki KH: Methodological issues of confounding in analytical epidemiologic studies. Caspian J Intern Med. 2012; 3(3): 488–495. PubMed Abstract \| Free Full Text\n\nRasmussen S, Shrestha S, Bjerregaard L, et al.: Antibiotic exposure in early life and childhood overweight and obesity: A systematic review and meta-analysis. Diabetes Obes Metab. 2018; 20(6): 1508–1514. PubMed Abstract \| Publisher Full Text\n\nAghaali M, Hashemi-Nazari SS: Association between early antibiotic exposure and risk of childhood weight gain and obesity: a systematic review and meta-analysis. J Pediatr Endocrinol Metab. 2019; 32(5): 439–45. PubMed Abstract \| Publisher Full Text\n\nAlonso-Coello P, Carrasco-Labra A, Brignardello-Petersen R, et al.: A methodological survey of the analysis, reporting and interpretation of Absolute Risk ReductiOn in systematic revieWs (ARROW): a study protocol. Syst Rev. 2013; 2: 113. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJohnston BC, Alonso-Coello P, Friedrich JO, et al.: Do clinicians understand the size of treatment effects? A randomized survey across eight countries. CMAJ. 2015; 188(1): 25–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeong KS, McLay J, Derraik JG, et al.: Associations of prenatal and childhood antibiotic exposure with obesity at age 4 years. JAMA Netw Open. 2020; 3(1): e1919681. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "68274", "date": "03 Aug 2020", "name": "Sepideh Soltani", "expertise": [ "Reviewer Expertise my research interest is systematic review and meta-analysis of observational and clinical trial studies." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Srivastava et al. examines the association between Early and frequent exposure to antibiotics in children and the risk of obesity. This is a well-conducted thorough review and meta-analysis. The literature search and the meta-analysis appear to have been conducted appropriately. The methods are well defined and the data appropriately statistically analyses. The figures and tables, including supplementary material, are appropriate. Overall the discussion is well written. There is no major concern about this manuscript.\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": "67985", "date": "10 Aug 2020", "name": "Bartłomiej M. Zalewski", "expertise": [ "Reviewer Expertise obesity", "evidence-based medicine" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe reviewers' objective was to evaluate evidence on the impact of prenatal, intrapartum, and early childhood antibiotic exposure and the association with the risk of overweight and obesity later in childhood.\nTo answer this question, the researchers decided to include in their systematic review both experimental and observational studies, however, only observational data was identified at this time. The Reviewers concluded that very low-quality evidence suggests that exposure to antibiotics early in life may be associated with an increased risk of overweight and obesity in later childhood.\nComments that should be addressed:\n\nGeneral comments:\n\n1: There are significant deviations from the PROSPERO protocol that should be commented on/explained:\ndifferent review question, different databases searched (less than pre-planned), different inclusion criteria (i.e. Protocol: the authors stated “Participants/population: Children aged 0 to 18, of any body weight and health status will be included.”; Manuscript: the authors did study the maternal prenatal and intrapartum antibiotic exposure.) two of the main outcomes (diabetes and change in the microbiome) were not reported at all. (If the authors did not find any studies we would recommend adding such a statement in the manuscript), different methods for the risk of bias assessment - what was the rationale for this change? pre-planned analyses of subgroups are different in the final paper than in the PROSPERO record [i.e. pre-planned analyses not done: subgroup analyses for boys versus girls; the effect of broad vs. narrow spectrum of antibiotics will be analyzed for all outcomes; we will assess the potential impact of early exposure (less than 3 years of age) to antibiotics versus later exposure (3 years of age or greater) (but done for 7y vs >=7y)].\n\nIn line with the above commentaries, the cited sentence should be changed accordingly (\"a priori design was published as a written protocol and registered with PROSPERO13. We closely followed our a priori protocol\").\n\n2a: In the methods section, the main outcomes of interest (overweight and obesity) are defined as BMI >25 kg/m2 for overweight and >30 kg/m2 for obesity, which is typically used in adults. It should be explained why the authors did not consider (in the methods) different, more appropriate outcomes for children definitions of overweight/obesity (i.e. >1SD for overweight and >2SD for obesity in children 5-19y in accordance to WHO). As most of the included studies were reporting the main outcome in percentiles – therefore the methods section should be changed accordingly.\n\n2b: In the PROSPERO protocol, pre-specified outcomes were 1. Overweight 2. Obesity. Please clarify what was behind the decision to group the second outcome as Overweight + Obesity? Why was the pre-planned outcome of \"obesity only\" not reported at all? There is at least a theoretical chance that the overweight effect was big enough to make a grouped outcome of overweight + obesity also significant, even if obesity alone was non-significant (alone). It would be of interest for all readers to complement this review with analysis for the obesity outcome only (as was pre-planned).\n\n3: As the literature search was conducted >24 months apart of completion of the review, and the Reviewers report other relevant studies in their discussion, one should consider updating the search, as following guidance from the Cochrane Handbook: “Reviews that are out of date and do not incorporate all the available evidence risk providing misleading information to decision makers and other stakeholders.” https://training.cochrane.org/handbook/current/chapter-iv.\n\nAdditional specific comments:\n\nAbstract:\n\nCurrently, the methods in the abstract do not match with those in the manuscript – please correct: (‘We conducted a comprehensive search of Embase, MEDLINE, and Web of Science for observational studies’ vs Inclusion criteria'. Observational and experimental study designs were eligible).\n\nResults section: could you add the number of participants eligible for both calculations of overweight and (overweight + obesity)?\n\nResults:\nThe flow diagram shows that the authors identified 12116 (12091+25). However, in the manuscript there is the number 12091 – please clarify.\n\nCould the authors provide the Table of Excluded studies?\n\nDoes the excluded studies table contain: Poulsen et al. (20171)? If not, what was the reason to exclude this study?\n\nTo increase readability, numerical references should be added in Table 1 near each included study.\n\nFigure 7 – not described adequately – low risk of bias?\n\nApart from the specific comments, we have additionally assessed this systematic review using the AMSTAR-2 critical appraisal tool (Shea et al., 20172) with an overall rating of confidence as moderate/low (due to deviations from the PROSPERO protocol and other non-critical weaknesses). \"No\" was answered to the following domains in the AMSTAR 2 tool: 2; 7,10;13.\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": "68273", "date": "13 Aug 2020", "name": "Xiuxia Li", "expertise": [ "Reviewer Expertise systematic review methodology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 conducted a systematic review and meta-analysis to evaluate evidence on the impact of prenatal, intrapartum, and early childhood antibiotic exposure and the association with the risk of overweight and obesity later in childhood. They concluded that very low-quality evidence suggests that exposure to antibiotics early in life may be associated with an increased risk of overweight and obesity in later childhood. This manuscript is well-written and the reporting quality is good. However, few edits should be made before indexing.\nThe authors should report any change/differences between the registered PROSPERO protocol and published manuscript. For example, different review question, different databases searched, different inclusion criteria and so on.\n\nPlease keep the inclusion criteria consistency about study design in the abstract section and those in the manuscript (‘We conducted a comprehensive search of Embase, MEDLINE, and Web of Science for observational studies’ vs Inclusion criteria ‘Observational and experimental study designs were eligible’).\n\nThe initial records (12091+25) in the flow diagram did not match with the in the 12091 in the results section. Please modify.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? Yes\n\nIs the statistical analysis and its interpretation appropriate? Yes\n\nAre the conclusions drawn adequately supported by the results presented in the review? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-711
https://f1000research.com/articles/9-710/v1	16 Jul 20	{ "type": "Research Article", "title": "Ablations over transformer models for biomedical relationship extraction", "authors": [ "Richard G Jackson", "Erik Jansson", "Aron Lagerberg", "Elliot Ford", "Vladimir Poroshin", "Timothy Scrivener", "Mats Axelsson", "Martin Johansson", "Lesly Arun Franco", "Eliseo Papa", "Erik Jansson", "Aron Lagerberg", "Elliot Ford", "Vladimir Poroshin", "Timothy Scrivener", "Mats Axelsson", "Martin Johansson", "Lesly Arun Franco", "Eliseo Papa" ], "abstract": "Background: Masked language modelling approaches have enjoyed success in improving benchmark performance across many general and biomedical domain natural language processing tasks, including biomedical relationship extraction (RE). However, the recent surge in both the number of novel architectures and the volume of training data they utilise may lead us to question whether domain specific pretrained models are necessary. Additionally, recent work has proposed novel classification heads for RE tasks, further improving performance. Here, we perform ablations over several pretrained models and classification heads to try to untangle the perceived benefits of each. Methods: We use a range of string preprocessing strategies, combined with Bidirectional Encoder Representations from Transformers (BERT), BioBERT and RoBERTa architectures to perform ablations over three RE datasets pertaining to drug-drug and chemical protein interactions, and general domain relationship extraction. We explore the use of the RBERT classification head, compared to a simple linear classification layer across all architectures and datasets. Results: We observe a moderate performance benefit in using the BioBERT pretrained model over the BERT base cased model, although there appears to be little difference when comparing BioBERT to RoBERTa large. In addition, we observe a substantial benefit of using the RBERT head on the general domain RE dataset, but this is not consistently reflected in the biomedical RE datasets. Finally, we discover that randomising the token order of training data does not result in catastrophic performance degradation in our selected tasks. Conclusions: We find a recent general domain pretrained model performs approximately the same as a biomedical specific one, suggesting that domain specific models may be of limited use given the tendency of recent model pretraining regimes to incorporate ever broader sets of data. In addition, we suggest that care must be taken in RE model training, to prevent fitting to non-syntactic features of datasets.", "keywords": [ "Natural Language Processing", "Biomedical Relationship Extraction", "NLP", "ChemProt", "Drug Drug Interactions", "Semeval 2010 Task 8" ], "content": "Introduction\n\nThe biomedical literature is a vast corpus of unstructured facts and findings, which need to be synthesised in some systematic way in order for drug discovery scientists to make informed, logical choices about what directions and experiments to pursue. A highly valued goal of biomedical natural language processing (NLP) is to perform relationship extraction (RE) between entities of interest1, such that the knowledge entombed within the literature can be exploited by technological solutions, such as knowledgebase representations. In recent years, groups such as BioCreative and SemEval have coalesced the community around shared RE tasks, in order that we might benchmark our methods against common standards.\n\nSince the early forays into transfer learning to the advent of transformer based models2,3, language modelling and, more recently, masked language modelling is the de rigour methodology in current NLP research. From investigations into the optimal learning objective, to explorations into the limit of pretraining, to permuting the classification head, a bewildering array of research has rapidly emerged, concerning almost every aspect of language modelling. This has created a vast experimental space for the community to explore how such developments relate to biomedical NLP.\n\nThe seminal masked language model, Bidirectional Encoder Representations from Transformers (BERT)4, helped to popularise the idea of pretraining on general linguistic data and subsequently fine tuning to tailor the model to downstream tasks. Pretraining is the task of learning some representation of language, such that a piece of text can be encoded into high dimensional space, representing some knowledge about how the tokens within such text relate to each other. Offshoots of BERT, such as SciBERT5, BioBERT6 and BlueBERT7, demonstrated that pretraining on scientific literature allow for better representations of the scientific sublanguage, leading to performance increases in downstream tasks pertaining to that domain. Work such as RoBERTa8 and T59 further recognised that BERT had been undertrained and built upon the original architecture with an expanded pretraining procedure and a larger parameter space.\n\nAlthough performance gains from larger models and lengthier pretraining is an interesting phenomenon, this represents practical issues for those working within niche domains who desire models pretrained on specific styles of document. With the rapid evolution of new architectures, and substantial costs involved in pretraining, the investment in performing domain specific pretraining becomes hard to justify when the end result may be obsolete within months. Thus, it is desirable to know whether the performance gains from domain specific pretraining outlive the original model architecture (compared to newer architectures that do not benefit from learning better representations of a domain, but perhaps benefit from learning better representations of domain independent, fundamental aspects of language).\n\nA second aspect of language modelling concerns how model are fine-tuned to perform certain tasks. For instance, sentence classification tasks with the original BERT model is possible by passing the sentence representation token (denoted [CLS]) through a linear layer. More recent work (specific to the task of relationship extraction) has explored how combining embedded entity information with such sentence representations can lead to significant performance boosts (the RBERT head)10. However, evidence has since emerged11 that at least some of the perceived performance gains of transformer style models is due to so-called ‘Clever Hans’ type effects, where the model is fine-tuned to learn unintended correlations in datasets rather than a generalised representation of the task. This in turn raises questions about the validity of such approaches in the task of relationship extraction, and how to manufacture appropriate datasets.\n\nThe goal of this article is to attempt to address some of these questions via ablation studies of a range of popular masked language models and classification heads, to determine their performance on the task of biological relationship extraction.\n\n\nMethods\n\nWe experiment with the general purpose pretrained BERT, model, the biomedical domain specific pretrained model, BioBERT, and the more recent general purpose RoBERTa model. Both BioBERT and RoBERTa are particularly relevant to the ablation tests in this study and serve as an example of a domain-specific model, and a larger model that has undergone lengthier pretraining, respectively. We combine these pretrained models with two classification heads; the commonly used linear layer based on the sentence vector produced by the final layer, and the RBERT classification head. In addition, we examine the effect of four string preprocessing techniques (two per classification head), to investigate how the differing transformer architectures respond to ablations.\n\nWe consider ablations over three different corpora labelled with named entities and relationships. The ChemProt12 dataset was originally created for the BioCreative VI workshop, and sought to challenge teams to deliver systems that extracted chemical protein relationships from the scientific literature. It consists of a set of 15,739 relationships annotations from 1,682 PubMed abstracts, divided into training, development and evaluation sets. The dataset covers 11 different label types, although only five undirected relationship types are used in the official evaluation. An official evaluation script is provided.\n\nThe DDI (Drug/Drug Interaction) corpus (hereafter DDI) was created for the SemEval-2013 DDI Extraction 2013 challenge13, and seeks to provide a dataset to support the development of NLP systems to extract various types of drug/drug interaction. It consists of 5,028 sentence level relationships manually annotated from Medline and DrugBank, labelled with one of five undirected classes (four describing different types of interaction and one null relationship class) and split into training and evaluation sets. The distribution of labels in this corpus is heavily weighted towards null relations, and thus the imbalance of classes represents an interesting problem for ML classifiers in its own right. The provided official evaluation script calculates the macro F1 over four relationship classes (the null relation is not considered).\n\nFinally, we make use of the Semeval 2010 Task 8 corpus14 (hereafter Semeval), which is a general English RE dataset collected from the Web, and uses a more abstract relationship classification schema than ChemProt or DDI. Here, ten relationship classes are used to annotate 10,717 sentences, which are split into a training set of 8,000 and an evaluation set of 2,717. The provided official evaluation script calculates the macro F1 over nine of the classes in a bi-directional fashion for a total of 18 classes.\n\nFor consistency with existing literature, we report our official scores (using official evaluation scripts provided with each dataset) but focus our analysis around cross validation on each set, in order to assess the consistency of the corpora and the effect of random seeds. Here, we report the mean macro averaged F1 score with a five-fold cross validation split.\n\nOriginally, we planned to conduct an analysis comparing a wide range of transformer architectures. However, our preliminary investigations suggested that many were too cumbersome to work with, either in terms of compute required, the quality of the pretrained model or the maturity of the codebase. To this end, we restricted our analysis to the pretrained models BERT Base, BioBERT 1.1, RoBERTa base and RoBERTa large, as described in Table 1. Our principal question compares the evaluation performance of BERT Base, BioBERT, and RoBERTa base, as models of approximately equal parameter counts. However, we additionally decided to include RoBERTa large to explore any potential benefits from using a larger model with a higher quality pretraining regime (based upon General Language Understanding Evaluation benchmark results15).\n\nAll experiments were conducted with the HuggingFace Transformers implementations, version 2.4.116\n\nPretrained models are frequently employed in classification tasks, wherein a linear layer is constructed on top of the final layer. Recently, some modifications of this approach have been proposed, to combine specific entity information into the classification layer, to support relationship classification tasks. Wu and He10 suggested averaging the token pieces representing each entity, and concatenating the output with the sentence vector before applying a fully connected feed forward layer, giving rise to the RBERT classification head and setting a new benchmark in the Semeval 2010 Task 8 dataset. In this work, we compare both the simple linear layer classification head and the RBERT head.\n\nRE is commonly construed as a sentence classification task, wherein the label assigned to the relationship between two entities in a sentence are instead assigned to the sentence. However, such an approach can be problematic; for instance, if there are more than two entities in a sentence, and/or more than two relationships (a common occurrence in biomedical text), leading to a situation where the same sentence can yield two conflicting labels.\n\nTo mitigate this, various strategies have been used, such as substituting the entities of interest with nominal placeholder tokens, such that all strings seen by a classifier are unique and creating the possibility for a classifier to learn the syntactic importance of the placeholder tokens with regard to the relationship that binds them17. In contrast, the RBERT architecture depends on inserting special characters around the two entities of interest, to inform the classifier of the two input entities without removing information about the entity itself.\n\nHere, we employ ablations on these preprocessing strategies depending on the type of classification head used with the pretrained model (Table 2).\n\nThe purpose of the sentence splitting ablation is to provide a baseline classification performance for the underlying pretrained model, without any special characteristics applied to the entities of interest (note, all other transformations include this sentence splitting step). The placeholder transformation is a commonly used strategy in RE6,18,19, where the entities in question are masked by some arbitrary token, thereby attempting to reduce overfitting of the classifier and allowing different relationships between different entity pairs in the same sentence to be represented. Similarly, the bounding special characters ablation is the original transformation as described in the original RBERT paper, whereas the purpose of the masked bounding special character transformation is to remove any entity specific information from the RBERT head. By removing this entity information, our intent is to explore the extent to which the positional information of the entity pairs are used in making the relationship classification, as opposed to the entity embedding information of the entity pairs.\n\nSince some of the preprocessing strategies can lead to undesirable mutations of the underlying data (for instance, it is not possible to represent discontinuous entity boundaries, or overlapping entity boundaries for the placeholder or bounding special character strategies), we filter out any such instances that cannot be transformed for all pretrained model/classification head configurations, such that our training and evaluation sets are consistent across all experiments.\n\nIn this ablation study, we aspire for consistency across experiments, rather than attempting to optimise for overall evaluation performance across our selected datasets. To this end, we do not attempt a hyper parameter search. Instead, we defer to recommended hyperparameters for classification tasks based upon the General Language Understanding Evaluation benchmark, as described in the original BERT and RoBERTa papers (Table 3).\n\nOne important consideration in hyperparameter selection is the maximum sequence length used. Naturally, it is desirable to use a sequence length big enough to enable the longest sentence in each dataset to be passed though the model. However, longer sequence lengths rapidly increase the memory usage in GPUs, and thus a variable batch size must be selected as required for a given dataset. Since larger batch sizes tend to be desirable20, we originally sought to specify a minimum batch size of 16 across all experiments, in line with recommendations in the BERT and ROBERTA papers. However, initial experiments uncovered that larger models such as RoBERTa large were unable to handle the required sequence length and batch size on the hardware available to us (Tesla V100 16Gb GPUs). To overcome this, we reduced the batch size to four and used eight gradient accumulation steps in all experiments.\n\nWe executed six runs across each dataset, per experiment configuration. The first run used the official train/test splits as described in the original datasets, whereas the remaining five runs were comprised of cross validation runs, varying the random seed between folds.\n\nWe trained for a maximum of five epochs, and after the first epoch, implemented an early stopping regime that tested for improvements in the average micro F1 score across all classes, after every 5% of the dataset. Five successive failures to improve the F1 resulted in the termination of training, and we logged the highest macro F1 scores reached during training for our cross validation results.\n\n\nResults and discussion\n\nThe results of each of our ablations are presented in Figure 1 (tabularised in Table 4).\n\nBERT_BC = BERT base cased, BERT_BIO = bioBERT, ROBERTA_B = RoBERTa base, ROBERTA_L = RoBERTa large, PH = placeholder, SSplit = sentence splitter, SpChar = bounding special characters, MSpChar = masked bounding special characters.\n\nBERT_BC = BERT base cased, BERT_BIO = bioBERT, ROBERTA_B = RoBERTa base, ROBERTA_L = RoBERTa large, PH = placeholder, SSplit = sentence splitter, SpChar = bounding special characters, MSpChar = masked bounding special characters. F1 values represents macro F1. Std = standard deviation for cross validation. Random token results use randomly ordered tokens in training data (evaluation data is kept intact).\n\nWith respect to the differences with BERT base cased and BioBERT, we observe a moderate benefit by using the BioBERT model on the biomedical Chemprot and DDI datasets, and a moderate benefit by using BERT base on SemEval, in line with observations that domain specific training can improve performance. However, BioBERT and RoBERTa large appear to be approximately equivalent across all datasets, with RoBERTa large ranking marginally higher in most experiments. The surprisingly poor performance of the RoBERTa base model compared to BERT base suggests that most of RoBERTa large’s performance is due to the higher parameter count, rather than the larger size of RoBERTa’s pretraining corpora. Nevertheless, given the very poor performance of the RoBERTa base model with the RBERT head, we are unable to rule out other factors. Particularly difficult to separate is the benefit of training on domain specific data; although RoBERTa is not a biomedical specific model, we examined the contents of the OpenWebText corpora upon which it is trained, and discovered over 11,000 references to PubMed abstracts, as well as other references to providers of scientific literature, suggesting that some of RoBERTa’s performance on biomedical text may come from partial exposure to the domain during pretraining.\n\nOn the biomedical datasets, the RBERT classification head seems to provide a small benefit in the DDI task, but this is not observed on the classification performance of ChemProt compared to the placeholder string transformation with the linear layer classifier head. However, the RBERT head appears to substantially boost performance on the SemEval dataset, although the benefits are substantially reduced if entity information is masked. In the case of SemEval dataset, this suggests that the classifier is making more use of the contextual entity embedding than the positional information of the token, and is therefore reliant on latent correlations between the entity pairs and the label, rather than an interpretation of the syntax of the sentence. In the case of the biomedical datasets, many of the classification head/string transformations performed similarly, suggesting none of these are particularly important and that the attention mechanism itself is mostly responsible for learning a representation of the data. A potentially related finding from our results is that even simple sentence classifiers give reasonable performance on the ChemProt and SemEval datasets, with no knowledge of what entity pairs in a sentence the label refers to. To explore this further, we randomised the token order for each instance in the training sets and repeated our experiments for the sentence splitter and placeholder string transformations (Figure 2). Although this ablation created a marked drop in performance across all datasets, we were surprised that this drop was not as substantial as we might have expected. By removing all syntactic information from the training data, it would appear (to a varying degree) that the classifiers are still able to learn some aspects of the relationship classification task using only contextualised embedding information.\n\nBERT_BC = BERT base cased, BERT_BIO = bioBERT, ROBERTA_B = RoBERTa base, ROBERTA_L = RoBERTa large, PH = placeholder, SSplit = sentence splitter. The horizontal blue line indicates the expected performance of a random classifier.\n\nWe suspect that this effect is likely to be attributed to the nature of the underlying training data. Although the attention mechanism employed by the models we tested should be able to learn the required syntactic relationships in order to perform the RE task21, it is also possible for them to learn other aspects of the training data that correlate the sentence embedding information with the given label. For instance, it seems likely that the the presence of certain words may occur more frequently with certain label types, such as verbs suggesting gene regulation activities in the case of ChemProt. Such an effect has recently been established for various NLP architecture across natural language inference datasets, including BERT22. Therefore, models that are trained to make use of such non-syntactic information probably generalise poorly, although further work will be required to establish this conclusively.\n\n\nConclusions\n\nIn this study, we perform a variety of ablations over an array of models and configurations over three RE datasets. We find that there are benefits in using models pretrained on biomedical text, but the benefits tend to be relatively small and/or task specific on the datasets we explored. Further, there is a tendency of newer models to be trained on larger corpora of text, which appear to encompass the biomedical domain. Future work might revisit analyses such as ours, to determine whether the benefits of domain specific model training outweigh the costs. Finally, we suggest that care must be taken in the training of models for RE, as it appears likely that classifiers are susceptible to overfitting on non-syntactic features. This may be alleviated by the creation of training data that depend heavily on syntactic features, and advancing other methodologies such as data augmentation and Universal Adversarial Triggers23.\n\n\nData availability\n\nThe DDI dataset is available from https://github.com/isegura/DDICorpus.\n\nThe ChemProt dataset is available from https://biocreative.bioinformatics.udel.edu/news/corpora/chemprot-corpus-biocreative-vi/.\n\nThe Semeval 2010 task 8 dataset is available from http://semeval2.fbk.eu/semeval2.php?location=data.\n\nSource code available from: https://github.com/RichJackson/pytorch-transformers\n\nArchived source code at time of publication: http://www.doi.org/10.5281/zenodo.389462524\n\nLicense: Apache 2.0", "appendix": "References\n\nHuang CC, Lu Z: Community challenges in biomedical text mining over 10 years: success, failure and the future. Brief bioinform. 2016; 17(1): 132–44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMalte A, Ratadiya P: Evolution of transfer learning in natural language processing. ArXiv. abs/1910.07370. 2019. Reference Source\n\nVaswani A, Shazeer N, Parmar N, et al.: Attention is all you need. NIPS. 2017. Reference Source\n\nDevlin J, Chang MW, Lee K, et al.: Bert: Pre-training of deep bidirectional transformers for language understanding. NAACL-HLT. 2019. Publisher Full Text\n\nBeltagy I, Cohan A, Lo K: Scibert: Pre-trained contextualized embeddings for scientific text. ArXiv. abs/1903.10676, 2019. Reference Source\n\nLee J, Yoon W, Kim S, et al.: Biobert: a pre-trained biomedical language representation model for biomedical text mining. Bioinformatics. 2019. PubMed Abstract \| Publisher Full Text\n\nPeng Y, Yan S, Lu Z: Transfer learning in biomedical natural language processing: An evaluation of bert and elmo on ten benchmarking datasets. BioNLP@ACL. 2019. Publisher Full Text\n\nLiu Y, Ott M, Goyal N, et al.: Roberta: A robustly optimized bert pretraining approach. ArXiv. abs/1907.11692. 2019. Reference Source\n\nRaffel C, Shazeer N, Roberts A, et al.: Exploring the limits of transfer learning with a unified text-to-text transformer. ArXiv. abs/1910.10683. 2019. Reference Source\n\nWu S, He Y: Enriching pre-trained language model with entity information for relation classification. CIKM ’ 19. 2019; 2361–2364. Publisher Full Text\n\nNiven T, Kao HY: Probing neural network comprehension of natural language arguments. In: Proceedings of the 57th Annual Meeting of the Association for Computational Linguistics. 2019; 4658–4664. Florence, Italy, Association for Computational Linguistics. Publisher Full Text\n\nKrallinger M, Rabal O, Akhondi SA, et al.: Overview of the biocreative vi chemical-protein interaction track. 2017. Reference Source\n\nHerrero-Zazo M, Segura-Bedmar I, Martínez P, et al.: The ddi corpus: An annotated corpus with pharmacological substances and drug-drug interactions. J Biomed Inform. 2013; 46(5): 914–20. PubMed Abstract \| Publisher Full Text\n\nHendrickx I, Kim SN, Kozareva Z, et al.: Semeval-2010 task 8: Multi-way classification of semantic relations between pairs of nominals. ArXiv. 2009. abs/1911.10422. Reference Source\n\nWang A, Singh A, Michael J, et al.: Glue: A multi-task benchmark and analysis platform for natural language understanding. BlackboxNLP@EMNLP. 2018. Reference Source\n\nWolf T, Debut L, Sanh V, et al.: Hug-gingface’s transformers: State-of-the-art natural language processing. ArXiv. 2019. abs/1910.03771. Reference Source\n\nLim S, Kang J: Chemical–gene relation extraction using recursive neural network. Database (Oxford). 2018; 2018: bay060. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDligach D, Miller T, Lin C, et al.: Neural temporal relation extraction. In: Proceedings of the 15th Conference of the European Chapter of the Association for Computational Linguistics: Volume 2, Short Papers. 2017; 746–751. Valencia, Spain, Association for Computational Linguistics. Reference Source\n\nShi P, Lin J: Simple bert models for relation extraction and semantic role labeling. 2019. Reference Source\n\nSmith LN: A disciplined approach to neural network hyper-parameters: Part 1 - learning rate, batch size, momentum, and weight decay. ArXiv. 2018. abs/1803.09820. Reference Source\n\nTenney I, Das D, Pavlick E: Bert rediscovers the classical nlp pipeline. ACL. 2019. Publisher Full Text\n\nThomas McCoy R, Pavlick E, Linzen T: Right for the wrong reasons: Diagnosing syntactic heuristics in natural language inference. ACL. 2019. Reference Source\n\nWallace E, Feng S, Kandpal N, et al.: Universal adversarial triggers for attacking and analyzing nlp. EMNLP/IJCNLP. 2019. Publisher Full Text\n\nWolf T, Debut L, SANH V, et al.: RichJackson/pytorch-transformers: supporting ablation paper v3. 2020. http://www.doi.org/10.5281/zenodo.3894625" }	[ { "id": "77522", "date": "03 Feb 2021", "name": "Yuan Li", "expertise": [ "Reviewer Expertise Biomedical natural language processing" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper compares the performance of a few pretrained models (BERT/BioBERT/RoBERTa x base/large) with different classification heads (Linear, RBERT) and pre-processing strategies (PH/SSplit for linear and SpChar/MSpChar for RBERT) on 3 relationship extraction datasets (2 biomedical and 1 general).\nThe results show that BioBERT is slightly better than BERT on 2 biomedical datasets but comparable to RoBERTa large on all 3 datasets. These observations can be explained because BioBERT is more domain-specific than BERT but the training corpus of RoBERTa also includes bio-related text such as PubMed abstracts and scientific literature. It is also shown that using RBERT head leads to a substantial improvement on the general dataset but not on two biomedical datasets. In addition, it is observed that models trained on randomly permuted data can still achieve better than random performance on all 3 datasets, which indicates that tokens themselves alone are still informative for the relationship extraction task.\n\nThis paper is well-structured and easy to follow. Although no new model is proposed, extensive experiments have been done comparing existing models in different settings. We appreciate that the code is provided, and that statistical variation is measured in the presented results.\nHowever, there are still a few concerns and places requiring clarification in the manuscript.\nThe reason for RoBERTa base’s poor performance using RBERT head is still unclear. The hyper-parameter settings from the original RoBERTa paper are used, but as we know that some hyper-parameters, like the learning rate, are associated with the size of mini-batch and input, it is worth doing a hyper-parameter search for different tasks. This may lead the authors to either find the cause of RoBERTa’s poor performance or exclude one possible source. Also, the authors could try to fine tune RoBERTa for a longer time, like 5 more epochs, to see if that will improve the performance.\nDue to the poor performance of RoBERTa base, it is better to include BERT large in all experiments. Since BERT base works reasonably well, a comparison between BERT base and BERT large can provide more meaningful insights, particularly in the context of a claim that larger models may offset the value of domain-specific tailoring.\nAlthough the authors state that their observation suggests that domain specific models may be of limited use given the tendency of recent model pretraining regimes to incorporate ever broader sets of data, it is hard to make blanket decisions based on this, like whether it is worthwhile to first pre-train a model on domain specific data for a specific task or directly use a pre-trained model. In practice, people may still need to try out both ways in specific contexts. Do the authors have more suggestions for practitioners?\nThe results of randomizing token order are not surprising because the relationship extraction task is defined as sentence classification and it is known that sentence classifiers that do not use any order information still work reasonably well in various NLP tasks 1,2]. This is because each word individually conveys a lot of information, unlike the case in computer vision where pixels are only meaningful when combined. Segment reordering has even been shown to be beneficial in certain tasks such as clinical Semantic Text Similarity 3, due to increasing robustness to linguistic variation. In addition, it is not a bad thing if a model can memorize training data that it has seen, and whether memorization will lead to poor generation is a separate question. So the authors should either provide evidence showing memorization will cause poor generalization in this case, or try out some methods mentioned in the conclusion section to improve the performance.\nApart from the above points, this paper also lacks a related work section which is important to contextualize the contributions of the work, in particular since some of the conclusions contradict prior results. Some missing references are listed below, and particularly relevant is 4, which comes to different conclusions. Although some of these have been published close to or even after the submission date of this paper, it would still be worth adding some discussion of them to a revision.\nPre-trained models other than BERT/BioBERT/RoBERTa: SpanBERT: Improving Pre-training by Representing and Predicting Spans (Transactions of ACL, 2020) KnowBERT -- Knowledge Enhanced Contextual Word Representations (EMNLP 2019) https://www.aclweb.org/anthology/2020.emnlp-main.379 https://www.aclweb.org/anthology/2020.clinicalnlp-1.17 Dedicated relation classification architecture: Downstream Model Design of Pre-trained Language Model for Relation Extraction Task (arXiv 2020)\nRelation classification as question answering: Relation Extraction as Two-way Span-Prediction (arXiv 2020)\nError analysis of relation extraction dataset and models: TACRED Revisited: A Thorough Evaluation of the TACRED Relation Extraction Task (ACL 2020)\n\nMinor comments: “knowledge entombed” -- We hardly think that the scientific literature is a tomb for knowledge. Perhaps find a different word. “this represents practical issues” → “this introduces practical issues”.\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": "93667", "date": "22 Sep 2021", "name": "Jens Dörpinghaus", "expertise": [ "Reviewer Expertise Biomedical natural language processing" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper is mostly dedicated to NLP and RE tasks within the biomedical field. Its main approach is to compare different pretrained models BERT, BioBERT and RoBERTa large/base in particular on drug-drug, chemical protein interactions and a general dataset. The authors conclude that there is not a great difference between these approaches, and suggest preferring pretrained models to domain specific models.\nThe paper is well-structured and sound. The authors provide not only references to their datasets, but also to their code. The contribution of this paper is limited to the comparison of different existing methods. This is my biggest concern: Since there is no section dedicated to the state of the art or related work, the decision on comparison is somehow unsubstantiated. I encourage the authors to provide this missing section and to provide more discussion on why they chose these models and what makes them particularly interesting. Some missing references as a starting point for further review include: Hoyt et al. (2021)1, Langnickel & Fluck (2021)2, Madan et al. (2017)3, and Yadav et al. (2020)4.\nThe conclusions are not totally convincing due to the fact that the method and analysis are not introduced well. Why is the analysis limited to these approaches? And why do they allow such a generalization? I doubt that the results allow suggesting a preference for pretrained models to domain specific models. Maybe the authors can provide more details on that.\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": [] } ]	1	https://f1000research.com/articles/9-710
https://f1000research.com/articles/8-263/v1	07 Mar 19	{ "type": "Research Article", "title": "Assessing the development and implementation of the Global Trigger Tool method across a large health system in Sicily", "authors": [ "Vincenzo Parrinello", "Elena Grasso", "Giuseppe Saglimbeni", "Gabriella Patanè", "Alma Scalia", "Giuseppe Murolo", "Peter Lachman", "Elena Grasso", "Giuseppe Saglimbeni", "Gabriella Patanè", "Alma Scalia", "Giuseppe Murolo", "Peter Lachman" ], "abstract": "Background: The Institute for Healthcare Improvement (IHI) has proposed a new method, the Global Trigger Tool (IHI GTT), to detect and monitor adverse events (AEs) and provide information to implement improvement. In 2015, the Sicilian Health System adopted IHI GTT to assess the number, types and severity levels of AEs. The GTT was implemented in 44 of 73 Sicilian public hospitals and 18,008 clinical records (CRs) were examined. Here we present the standardized application of the GTT and the preliminary results of 14,706 reviews of CRs. Methods: IHI GTT was adapted to the local context, and developed and implemented. Reviews of CRs were conducted by 199 professionals divided into 71 review teams consisting of three individuals: two of whom had clinical knowledge and expertise, and a physician to authenticate the AE. The reviewers entered data into a dedicated IT-platform. All 44 of the public hospitals were included, with approximately 300,000 inpatient yearly admission out of a population of approximately 5 million. In total, 14,706 CRs of inpatients from medicine, surgery, obstetric and ICU wards, from June 2015 to June 2018 were reviewed. Results: In 975 (6.6%) CRs at least one AE was found. Approximately 20,000 patients of the 300,000 discharged each year in Sicily have at least one AE. In 5,574 (37.9%) CRs at least one trigger was found. A total of 1,542 AEs were found. The analysis of ROC curve shows that the presence of two triggers in a CR indicates an AE with a high probability. The most frequent type of AE was in-hospital related infection. Conclusions: The GTT is an efficient method to identify AEs and to track improvement of care. The analysis and monitoring of some triggers is important to prevent AEs. However, it is a labor-intensive method, particularly if the CRs are paper based.", "keywords": [ "Global Trigger Tool", "patient safety", "adverse events detections", "quality of care", "medical errors", "harm" ], "content": "Introduction\n\nSafety is one of the domains of quality in healthcare. Improving the safety of patients is a political priority worldwide, as studies on the safety of patients have drawn attention to the high rates of health care-related harm1–3. Improving patient safety requires effective and reliable methods to identify and monitor adverse events (AEs) so that learning can take place and improvements can be made.\n\nEven though several methods to detect AEs are available, there is no universally recognized method that reliably provides a comprehensive overview of the extent of the problem. These methods include incident reporting, clinical records (CRs) review and automated extraction using hospital administrative data, for example Patient Safety Indicators (PSI) as developed by the Agency for Healthcare Research and Quality (AHRQ). Incident reporting is the most commonly used method to detect AEs in hospitals, but is based on voluntary reporting. Despite considerable efforts by local hospitals, reporting systems only detect a limited number of AEs4. The effectiveness of automated extraction using hospital administrative data for detecting AEs depends on the accuracy of data compilation. CRs review, as used in the Harvard Medical Practice Study, is very labor intensive, thereby limiting its use4. As a result, health services, governments and researchers have focused on developing harm detection tools.\n\nThis paper is one of the first to report the findings of the application of the Global Trigger Tool, developed by the Institute for Healthcare Improvement (IHI), across the whole health system in Sicily. In 2015, the Sicilian Health System adopted IHI GTT5 to assess the number, types and severity levels of AEs.\n\n\nMethods\n\nThe measurement instrument used in our study is the Italian version of the IHI GTT6. The Italian version was adapted to be appropriate to the regional context7. The triggers are grouped into the same seven categories as the original version (care, medication, surgical, intensive care, obstetric, pediatrics and emergency care); however, changes to some triggers have been introduced. We did not consider triggers and AEs that were present on admission and we added three new triggers: change in procedure anesthesia, duration of surgery greater than 6 hours, and hospital stay greater than five days after delivery7.\n\nThe sampling method followed that recommended by the IHI GTT, with 10 inpatient CRs randomly selected monthly. From the ordered sequence of the numbering of the CRs of the period under evaluation, a CR was selected every 10 (ie 10th, 20th, 30th, 40th, 50th, etc). In the case in which the number of patients discharged during that month is less than 100, we proceed to remove the previously selected CRs and selecting another CR in the same way (one CR every 10). Eligibility criteria were an admission lasting more than 24 hours, and all the administrative data completed. In the Intensive Care Unit (ICU), all patient CRs, discharged during the reference period, were reviewed.\n\nAs per the IHI protocol, each review team was composed of three individuals: two with clinical knowledge and expertise on patient clinical documentation, and a physician whose role was to authenticate the findings and the severity rating of the AEs. The total number of the reviewers was 199 divided into a 71-person team. Where possible, the review team remained consistent over time.\n\nWe excluded triggers and/or events that took place outside the time of the patient admission to the hospital and we considered only triggers and AEs that occurred during hospitalization. The two clinical reviewers audited all the CRs on their own. We used five worksheets: general care, medication, surgical, obstetric and intensive care, with some changes in accordance with the IHI GTT. The CRs were examined following the order of the sections described in the IHI GTT. Limit for review of each patient record was 20 minutes. The “20-minute rule” was applied to all records regardless of size5. The reviewers entered data into a specially developed dedicated IT-platform, developed by our IT team (based on Jawascript HTML and PHP)8\n\nFor the statistical analysis we used the software SPSS ver. 20. We used it also to develop the Receiver Operating Characteristic (ROC) curve analysis.\n\n\nResults\n\nFrom June 2015 to June 2018, 18,008 CRs from 105 wards of 44 Sicilian public hospitals were examined. In this study, we analyzed 14,706 CRs relating to patients discharged from 89 medicine, surgery, obstetrics and intensive care wards. In 5,574 (37.9%) CRs at least one trigger was found. In 7 CRs an AE was detected without a trigger being present. AEs were determined in 1,542 CRs (Table 1). The identification of triggers allowed us to identify corresponding AEs (Table 2).\n\nCRs, clinical records; ICU, intensive care unit; AES, adverse events\n\nAEs, adverse events.\n\nThis analysis allowed us to highlight how isolated triggers are not always a good indicator of AEs. A Receiver Operating Characteristic (ROC) curve analysis demonstrates that the presence of two triggers in a CR has a high probability of an AE having occurred (Figure 1). In CRs with a high frequency of triggers, a corresponding number of AEs was not always detected. As indicated in Table 3, on the contrary, some triggers were associated with a large number of AEs. Triggers and AEs were analyzed when isolated triggers were identified (Table 4). For example, the isolated trigger C01 (Blood products use) was present in 483 cases, but identified only two AEs, and the trigger M05 (Rising BUN or serum creatinine >2 times the baseline) did not identify any AEs. AEs were classified using the 2009 edition of the WHO International Classification for Patient Safety (ICPS)9, and a clinical classification developed by our group (Table 5).\n\nROC curve shows that the presence of two triggers in clinical records indicates an adverse event with a high probability.\n\nAEs, adverse events.\n\nAEs, adverse events.\n\nAEs, adverse events.\n\nThe most frequent type of AEs observed: in-hospital related infections; surgical complications; pressure ulcers; acute kidney injury; and procedure complications.\n\n\nDiscussion\n\nThe evaluation of the quality and safety of health systems is difficult, but has become a priority of healthcare funders and organizations. Outcome, management and patient satisfaction indicators are available to measure the different dimensions of health care quality, but reliable measurements of safety have been elusive. Many methodologies and indicators, such as the PSI developed by AHRQ, and the review of health documentation and incident reporting are currently used. The documentation and study of AEs, i.e. where they occur, and the type and degree of harm, is essential to promote specific opportunities for improvement interventions and to evaluate effectiveness of any intervention over time.\n\nThe IHI GTT is one methodology proposed to detect and monitor AEs and provide information to implement improvement. At present, compared to other methods, it may be the best methodology to use4. A systematic review reported the use of GTT methodology in 15 countries in 44 hospitals, with 79,004 clinical records examined10. The data are an underestimation, as the report did not include some comprehensive Swedish and Norwegian studies11. Recently, papers have been published in Italy, Austria, China and Russia12–16. A critical appraisal of the studies and their results is difficult, as the methodology used is heterogeneous, protocols are often locally adapted to the local context, the populations studied are different, and the skills of the reviewers vary. We adapted the IHI GTT to the local context in Sicily for this study and did not consider triggers and AEs identified at the admission of the patient as well as modifying some triggers. In this study, the triggers were analyzed both when associated with other triggers and when isolated. In both cases the correlation with AEs was analyzed.\n\nIn this study, 37.9% (n=5,574) of CRs examined had triggers and in 18.9% (n=2,778) an isolated trigger was found. CRs with triggers (n=5,574) are significantly present in surgery wards (n=1,709; 35.4%), medicine wards (n=1,517; 34.3%) and ICU (n=1,672; 84.7%). while CRs with triggers in obstetrics wards are significantly less frequent (n=676; 20.3%). CRs with isolated triggers are more common in medical wards (n=1,085; 23.7%) and rarer in ICU (n=272; 13.7%) (Table 1).\n\nThe connection between the number of CRs with triggers and the number of CRs examined is not always reported in the literature and when reported it is not always clear. Xu et al.17 report that during review of 240 clinical records, 51.0% triggers (26/51) were identified 206 times. Mortaro et al.12 report that during review of 1,320 clinical records, a total of 130 triggers were detected.\n\nIn general, it would appear that little attention is paid to triggers if they are not related to an AE. Instead, many triggers of the IHI GTT protocol could be considered to be a measure of near misses and potential AEs. These include, decrease of Hb or Ht >25%, readmission within 30 days, transfer to higher level of care, clostridium difficile–positive stool, PTT >100 seconds, INR > 6, glucose < 50 mg/dl, rising BUN or serum creatinine >2 times baseline, blood loss >500 mL (after vaginal delivery) or >1000 mL (Cesarean section), and readmission to ICU.\n\nIn a systematic review, de Vriess et al.18 reported that in 8 studies that included 74,485 CRs, the median overall incidence of in-hospital AEs was 9.2%. Another systematic review reported 44 hospitals with 79,004 CRs, had an incidence between 7 and 51%10. In the Sicilian public hospitals, 1,542 AEs were detected in 975 clinical records, corresponding to an incidence of 6.6% of CRs examined, and to 17.5% of CRs with triggers. It would appear that the percentage of clinical records with triggers increases with the increase of percentage of CRs examined, compared to the patients discharged, while the percentage of CRs with AEs remains more stable (Figure 2).\n\nICUs have the highest incidence of AEs, both with respect to CRs examined (30.4%) and those with triggers (35.9%) (Table 1). This could be due to patients being transferred to ICU and the cause of the AE was in another clinical setting. We analyzed the AEs comparing them to the triggers to allow for their identification:\n\n￭ Most AEs are associated with general care triggers (n=6,103) (Table 2). If triggers are isolated, AEs are more frequently associated with care triggers (n=80) (Table 4).\n\n￭ The triggers related to general care have been identified 7,497 times, with an AE in 6,103 (81.4%).\n\n￭ Medications-related triggers have been identified 2,878 times, with an AE in 1,525 (53%).\n\n￭ Surgery-elated triggers have been identified 323 times, with an AE in 148 (45.8%).\n\n￭ Obstetrics-related triggers have been identified 492 times, with an AE in 61 (12.4%).\n\n￭ Intensive-care-related triggers have been identified 1,817 times with an AE in 1,924 (i.e. it is very common for triggers to identify more AEs in the same patient) (Table 1 and Table 2).\n\nHowever, if isolated triggers are considered, the intensive-care-related triggers were detected 46 times and they were correlated with only one AE (Table 4). This observation suggests that isolated triggers rarely allow to identify an AE and that the strength of the IHI’s GTT methodology is linked to the association of triggers. It is evident that the detection of many triggers in a CR is associated with a high probability of AEs. The analysis of the ROC curve (Figure 1) shows that it is sufficient to detect two triggers in a CR because it can be almost certain that in that CR there may be an AE.\n\nWe have classified the AEs using the ICPS 2009 classification and a clinical classification, developed by our group. In both classifications, hospital acquired infections are the most frequent AEs present (Table 5), observed in the ICU in 625 clinical records (84.3%). Surgical complications (n=175) were observed in 55.9% (n=99) in ICU, i.e. they were AEs in patients undergoing surgery and then transferred to ICU due to the onset of a complication. In 44.6% (n=80), the AEs are represented by hemorrhagic complications (intra- and post-operative hemorrhages or hematomas). Pressure ulcer lesions were detected in 172 cases, usually in the ICUs (n=112 - 65.1%). Also, the complications from procedures (n=109) were observed mainly in the ICU (n=78; 71.5%). In total, 33 complications from procedures are related to orotracheal intubation, 22 at central venous catheter, and 10 at childbirth analgesia. The complications of child birth (n= 47) were represented more frequently in 59.5% (n=28) by bleeding and in 29.8% (n=14) by lacerations.\n\nOur study has some several limitations. The first concerns the inter-rater reliability assessment of review teams that is not available. The second limitation is the underlying quality of CRs, which may have affected the results. However, all reviewers received the same training and each team followed the same protocols to ensure reliability.\n\n\nConclusion\n\nThe Global Trigger Tool is an effective method to identify AEs and track improvement of care. It provides to clinical teams an understanding of the patient safety issues that are present in their clinical area, as well as opportunities to improve. With active involvement of clinical teams, it places patient safety in the centre of clinical activity and fosters a culture of safety. It also provides an effective way to assess the quality of the clinical records. The drawback is that the process is labor intensive, particularly if the clinical records are paper based. The introduction of electronic medical records would allow a quicker process with the automation of the identification of triggers and the possibility to link triggers together in the identification of adverse events and near misses, especially where there has been more than one trigger19. Finally, we conclude that the analysis and monitoring of some triggers, as potential indicators of near misses, is important to prevent adverse events.\n\n\nEthical considerations\n\nSince the data used in this study was gathered during routine practice and is used for analysis of hospital procedures, no ethical approval was obtained. Every patient gave written informed consent on admission to hospital for the use of their data for scientific research. This consent is the \"Information on the processing of personal data\" with reference to the Italian law n. 196/2003 and Regulation (EU) 2016/679 of the European Parliament and of the Council of 27 April 2016 (UE).\n\n\nData availability\n\nHarvard Dataverse: Replication data for Developing and implementing the Global Trigger Tool methodology across a large health system in Sicily, https://doi.org/10.7910/DVN/YQNKCC20\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).", "appendix": "Grant information\n\nThis work is supported by Italian Healthcare Ministry and Sicilian Healthcare Ministry (PSN1316.3).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nIt was possible to realize this project thanks to the involvement and passion of the 199 reviewers, doctors, nurses and obstetrics, the cooperation of quality and patients safety managers of the Sicilian public hospitals, Antonino Drago and Rosario Raineri for their support on data analysis.\n\n\nReferences\n\nLeape LL, Brennan TA, Laird N, et al.: The nature of adverse events in hospitalized patients. Results of the Harvard Medical Practice Study II. N Engl J Med. 1991; 324(6): 377–384. PubMed Abstract \| Publisher Full Text\n\nLocalio AR, Lawthers AG, Brennan TA, et al.: Relation between malpractice claims and adverse events due to negligence. Results of the Harvard Medical Practice Study III. N Engl J Med. 1991; 325(4): 245–251. PubMed Abstract \| Publisher Full Text\n\nBrennan TA, Leape LL, Laird NM, et al.: Incidence of adverse events and negligence in hospitalized patients. Results of the Harvard Medical Practice Study I. N Engl J Med. 1991; 324(6): 370–376. PubMed Abstract \| Publisher Full Text\n\nClassen DC, Resar R, Griffin F, et al.: 'Global trigger tool' shows that adverse events in hospitals may be ten times greater than previously measured. Health Aff (Millwood). 2011; 30(4): 581–9. PubMed Abstract \| Publisher Full Text\n\nGriffin FA, Resar RK: IHI Global Trigger Tool for Measuring Adverse Events. 2nd ed. (IHI Innovation Series white paper). Cambridge, MA: Institute for Healthcare Improvement. 2009. Reference Source\n\nMoretti F, Mortaro A, Pascu D, et al.: Uno strumento per la quantificazione degli eventi avversi: il Global Trigger Tool dell'Institute for Healthcare Improvement (IHI). 2013; (Accessed 7th November 2018). Reference Source\n\nAzienda Ospedaliero-Universitaria “Policlinico-Vittorio Emanuele, Catania. U.O. per la Qualità e Rischio Clinico. Il protocollo di rilevazione. Reference Source\n\nAssessorato regionale della salute. Regione Siciliana. IT platform. Reference Source\n\nConceptual Framework for the International. Classification for Patient Safety. Version 1.1. Final Technical Report. 2009. (Accessed 7th November 2018). Reference Source\n\nHibbert PD, Molloy CJ, Hooper TD, et al.: The application of the Global Trigger Tool: a systematic review. Int J Qual Health Care. 2016; 28(6): 640–649. PubMed Abstract \| Publisher Full Text\n\nNilsson L, Borgstedt-Risberg M, Soop M, et al.: Incidence of adverse events in Sweden during 2013-2016: a cohort study describing the implementation of a national trigger tool. BMJ Open. 2018; 8(3): e020833. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMortaro A, Moretti F, Pascu D, et al.: Adverse Events Detection Through Global Trigger Tool Methodology: Results From a 5-Year Study in an Italian Hospital and Opportunities to Improve Interrater Reliability. J Patient Saf. 2017. PubMed Abstract \| Publisher Full Text\n\nDeilkås E, Bukholm G, Lindstrøm JC, et al.: Monitoring adverse events in Norwegian hospitals from 2010 to 2013. BMJ Open. 2015; 5(12): e008576. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoffmann-Völkl G, Kästenbauer T, Mück U, et al.: [Detection of adverse events using IHI Global Trigger Tool during the adoption of a risk management system: A retrospective study over three years at a department for cardiovascular surgery in Vienna]. Z Evid Fortbild Qual Gesundhwes. 2018; 131–132: 38–45. PubMed Abstract \| Publisher Full Text\n\nJi HH, Song L, Xiao JW, et al.: Adverse drug events in Chinese pediatric inpatients and associated risk factors: a retrospective review using the Global Trigger Tool. Sci Rep. 2018; 8(1): 2573. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nФастовец МН, Белорус АИ, Лысак ВП, et al.: [Incidence of adverse medical events in the neonatal intensive care unit with the help of a global trigger tool]. Wiad Lek. 2017; 70(3 pt 1): 483–488. PubMed Abstract\n\nXu XD, Yuan YJ, Zhao LM, et al.: Adverse Events at Baseline in a Chinese General Hospital: A Pilot Study of the Global Trigger Tool. J Patient Saf. 2016. PubMed Abstract \| Publisher Full Text\n\nde Vries EN, Ramrattan MA, Smorenburg SM, et al.: The incidence and nature of in-hospital adverse events: a systematic review. Qual Saf Health Care. 2008; 17(3): 216–223. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMevik K, Hansen TE, Deilkås EC, et al.: Is a modified Global Trigger Tool method using automatic trigger identification valid when measuring adverse events?: A comparison of review methods using automatic and manual trigger identification. Int J Qual Health Care. 2018. PubMed Abstract \| Publisher Full Text\n\nParrinello V: \"Replication data for to Developing and implementing the Global Trigger Tool methodology across a large health system in Sicily\". Harvard Dataverse, V1. 2019. http://www.doi.org/10.7910/DVN/YQNKCC" }	[ { "id": "45400", "date": "28 Mar 2019", "name": "James M. Naessens", "expertise": [ "Reviewer Expertise Health Services Research", "Patient Safety and Quality of Care Measurement", "Biostatistics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper by Parrinello et al., presents the experience of applying the Italian version of the Institute for Healthcare Improvement’s Global Trigger Tool (GTT) for screening for adverse events (AEs) to a random sample of medical records for hospital discharges from June 2015 to June 2018 for all 44 public hospitals in Sicily. The authors have done a nice job of describing their overall experience with the GTT and focused on the quantity and type of triggers which helped identify adverse events. There were a few issues where some further explanation would clarify the methods and results, and aid the reader in evaluating the contributions of this paper. In addition, there were a couple of minor editing points:\nIn my first reading of the paper, I was confused by Table 2. It is not clear how a trigger could be associated with AEs more often than it is detected. This happened with C03, C04, C08, etc. Table 3 presents some of the same information, but labels the second column as AEs with triggers. To evaluate the effectiveness of GTT as a screening tool, it is useful to see both what percent of records with a specific trigger had an AE as well as how many AEs were associated with that trigger. The authors should clarify in Methods how they identified AEs (role of physician, why record without a trigger was reviewed) and clarify whether results they present are based on the count of AEs or the percent of records with AEs (or both). I’d recommend both, as in Table 1.\n\nThe first paragraph of the results states that 18,008 records were reviewed, but only 14,706 were analyzed. The authors should explain why the records were excluded, to show they did not bias the results. A flow chart might help. They also state that 7 CRs had AEs without triggers. My understanding of the GTT method is that only records with triggers are reviewed for AEs. Please explain how these AEs were found. The first paragraph under “Rates of triggers” in the discussion, which should be moved to the Results section, refers to significant differences. The method of testing these statistics should be added to the “Statistical analysis” section of the Methods.\n\nThere were several typos or minor errors:\nP3, Methods, Review team: 71-person team (?). Table 1, lines 2 vs. 4 and lines 3 vs. 5: not clear what the differences are. How do we get different %’s? Figure 1: style of sensitivity line in graph does not match that of label. Table 5: Procedure complication has an asterisk, but no explanation. Figure 2: typo (EA) in 3rd label. P9: surgery-elated (?).\n\nAfter reading the manuscript, it appears that there are two main questions that can be answered from the results:\nHow does patient safety in Sicily, based on the GTT, compare to other published studies? Can we learn anything from the analysis of the triggers that can help us improve the review process?\nThe first question is addressed in the discussion (page 4). Adding a confidence interval to the reported rate from this study (6.6%) would clarify that these hospitals are as good or better than other reported rates.\nThe most important conclusions I would draw from the trigger analysis is that few AEs were identified by isolated triggers and many isolated triggers are not associated with AEs. Although not highly useful for identifying AEs, some isolated triggers may be direct measures of “near misses”. I would restructure the results and discussion to emphasize these points.\nOverall, this is a good description of a broad-based standardized implementation of the Global Trigger Tool. The authors have presented suggestions for making improvements to the method.\n\nIs the work 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? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4533", "date": "11 Sep 2019", "name": "vincenzo parrinello", "role": "Author Response", "response": "We appreciate the review by Professor Naessens of our paper, the accuracy of his observations and the relevance of his suggestions. We are honored that he considers our paper “a good description of a broad standardized implementation of the Global Trigger Tool” and that our work “presented suggestions for improving the method.” We are aware that some of the points require further clarity, which may have been lost as we limited the word count. CommentIn my first reading of the paper, I was confused by Table 2. It is not clear how a trigger could be associated with AEs more often than it is detected. This happened with C03, C04, C08, etc. Table 3 presents some of the same information, but labels the second column as AEs with triggers. To evaluate the effectiveness of GTT as a screening tool, it is useful to see both what percent of records with a specific trigger had an AE as well as how many AEs were associated with that trigger. The authors should clarify in Methods how they identified AEs (role of physician, why record without a trigger was reviewed) and clarify whether results they present are based on the count of AEs or the percent of records with AEs (or both). I’d recommend both, as in Table 1.ResponseTable 2 In the third column of this table we reported the number of times a trigger was found.In the fourth column the number of times in which an adverse event was related to that trigger.The title of the fourth column (Times associated, with AE)does not express this clearly.Therefore in the third column the triggers are indicated, while in the fourth column the adverse events are indicated. We have found some CRswith more than one adverse event. In the third column of table 2 we intended to report that the C03 trigger was found in 279 CRs examined. Instead, in the fourth column of Table 2 we wanted to indicate that in 438 adverse events the C03 trigger was present.Table 3We have tried to elucidate this concept in Table 3.This table lists the triggers that, in our experience, require more focus on. Finding one of the triggers on this list, linked to other triggers, could indicate the presence of more than one adverse event.Effectiveness of a GTTWe agree that to evaluate the effectiveness of the GTT as a screening tool, it is useful to see both what percentage of CRs with a specific trigger have an AE and how many AEs have been associated with that trigger. We have a table to explains this, but due to space constraints it was not included and we can add it if the editorial staff agree.Definitions and processAdverse events were identified based on the IHI protocol definition: \"unintended physical injury resulting from or contributed to medical care that requires additional monitoring, treatment or hospitalization, or that results in death.\" The physician reviewed the consensus with the two records and reached a final agreement on the type, number, and severity of events. The physician did not review the CRs, only the summary sheet in IT-platform.Table 1Table 1 shows the number of CRs examined, the number of CRs with triggers and the percentage of discharged patients. One line is missing which provides the number of discharged patients.If the editorial staff allows, we can integrate Table 1 with the number of patients discharged and the number of AEs compared to the CRs examined and with triggers. CommentThe first paragraph of the results states that 18,008 records were reviewed, but only 14,706 were analyzed. The authors should explain why the records were excluded, to show they did not bias the results. A flow chart might help.ResponseFrom June 2015 to June 2018, 18.008 CRs were examined. In this study, we analyzed 14,706 CRs relating to patients discharged from 89 medicine, surgery, obstetrics and intensive care wards. 3.302 CRs concerned pediatrics and emergency department wards. Comment They also state that 7 CRs had AEs without triggers. My understanding of the GTT method is that only records with triggers are reviewed for AEs. Please explain how these AEs were found.ResponseExamination of the CRs was performed in accordance with the IHI protocol: Discharge codes, particularly infections, complications, or certain diagnoses Discharge summary Medications administration record Laboratory results Prescriber orders Operative records (operational report and record anesthesia, if applicable) Nursing notes Physician progress notes If time permits, any other areas of the record In seven CRs the reading of the Discharge codes and of the Discharge summary has allowed us to identify directly the adverse events represented by two cases of invasive procedures complications, two cases of surgical complications, a case of hypoglycemia and a case of adverse reaction to the administration of a drug. Therefore the reviewers, having identified the adverse event, did not look for triggers. Comment The first paragraph under “Rates of triggers” in the discussion, which should be moved to the Results section, refers to significant differences. The method of testing these statistics should be added to the “Statistical analysis” section of the MethodsResponse The term \"significantly\" used in the first paragraph under \"Rates of triggers\" in the discussion, is misleading. We did not intend to claim that there was a statistically significant difference. CommentStyle of sensitivity line in graph does not match that of label of Figure 1.Response if possible we will correct the figure. Comment P3, Methods, Review team: 71-person team (?).Response The total number of reviewers was 199 divided into 71 teams.In some teams, medical reviewers from another team were the supervisors. Comment Table 1, lines 2 vs. 4 and lines 3 vs. 5: not clear what the differences are. How do we get different %’s?ResponseIn lines 2 and 4 of table 1 the number of CRs with triggers are shown.In line 2, the % refers to the total number of patients discharged; in line 4 the % refers to CRs with triggers. For example, from June 2015 to June 2018, 1,571 medical cases with triggers were found in the medical departments, equal to 34.3% of all patients discharged in the same period (n = 4,527) and 33.4% of all triggered CRs (n = 5,574). I think it would be clearer if we put a line with the number of patients discharged. Comment Table 5: Procedure complication has an asterisk, but no explanation.Response The explanation is Central Venous Catheter (CVC), Continuous Veno-Venous Hemofiltration (CVVH), Endoscopy, Orotracheal Intubation, Endoscopic Retrograde Cholangiopancreatography (ERCP).If possible we will correct the table. Comment Figure 2: typo (EA) in 3rd label.Response Figure 2: typo (EA) in 3rd label. is a translation error (AE). In Italian it is EA, Comment P9: surgery-elated (?).Response it would be surgery-related. Comment After reading the manuscript, it appears that there are two main questions that can be answered from the results: How does patient safety in Sicily, based on the GTT, compare to other published studies? Can we learn anything from the analysis of the triggers that can help us improve the review process? The first question is addressed in the discussion (page 4). Adding a confidence interval to the reported rate from this study (6.6%) would clarify that these hospitals are as good or better than other reported rates.ResponseRegarding the addition of a confidence interval at the rate reported by this study (6.6%) to clarify if these hospitals are equal or better than other reported rates, we think that the methodologies used in the various studies are too heterogeneous, protocols are often locally adapted to the local context, the populations studied are different, and the skills of the reviewers vary. For these reasons we decided not to explore this topic from a statistical point of view. CommentThe most important conclusions I would draw from the trigger analysis is that few AEs were identified by isolated triggers and many isolated triggers are not associated with AEs. Although not highly useful for identifying AEs, some isolated triggers may be direct measures of “near misses”. I would restructure the results and discussion to emphasize these pointsResponseWe agree and we will emphasize this point." } ] }, { "id": "46773", "date": "01 May 2019", "name": "Brent C. James", "expertise": [ "Reviewer Expertise Clinical quality improvement", "patient safety", "high-reliability organizations" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Harvard Medical Practice Study (HMPS – cited by the authors) used post-discharge chart review to detect care-associated injuries (adverse events – AEs) that occurred during hospitalization. Under HMPS, trained nurses reviewed a random selection of charts. If those nurses discovered what they judged to be care-associated injuries, they flagged those events in the chart as a potential AE. Charts that contained one or more AEs were forwarded to 2 independent physician reviewers. If both physician reviewers judged, for each AE, that event had occurred, then it was reported as a confirmed AE. Those physicians also judged whether each AE was avoidable, and whether each could be considered negligent (substandard) care.\nThe IHI Global Trigger Tool (IHI GTT) built on the HMPS methodology. It attempted to improve the ability of initial nurse reviewers to detect potential AEs, by providing a set of 51 “review triggers,” falling into 7 major subcategories – explicit initial events that, if detected in the chart, chained to examination of other specific events. Initial assessments of the IHI GTT showed much higher detection rates for AEs than did the original HMPS methodology, which itself far exceeded typical voluntary reporting mechanisms.\nThis study reviews the use of the IHI GTT in 44 Sicilian public hospitals across a 3 year time period – June 2015, through June 2018. The study’s authors adapted the IHI GTT to their specific environment. They also added 3 additional review triggers, beyond those included in the original IHI GTT. They focused their analysis on a subset of all charts assessed with their modified IHI GTT: They analyzed only charts for patients hospitalized on medicine, surgery, obstetrics, and intensive care wards.\nAs this study notes, many other groups are using the IHI GTT to detect AEs. This work is useful because it supplies empiric observation of one such use, across a large system of hospitals and an extended period of time.\nThis report has at least 2 major differences from other work in the field. First, only 6.6% of records reviewed had at least one care-associated adverse event. Other studies have shown much higher AE rates. The authors note this in their text, and suggest it may reflect differences in local environments and chart review methods. Second, most other IHI GTT reviews show adverse drug events (medication-related events - overdoses, drug-drug interactions, and allergic or idiosyncratic reactions) as the dominant category of AEs detected. In this study, they are a distant number 3. Why?\n\nSpecific suggestions:\nIt appears that the entire IHI GTT program assessed a total of 18,008 records from among 105 hospital wards from June 2015 through June 2018. However, this analysis examines only medicine, surgery, obstetrics, and intensive care wards. Those represented a total 89 wards, with a total of 14,706 records reviewed.\nThis is unclear in both your abstract and your text. It would be very helpful to more clearly explain how you derived the CRs included in your analysis.\n\nYou mention that you had a total of 199 individuals who participated in 71 3-person review teams. Obviously, some people participated in more than 1 team. It is not clear how the 2 initial reviewers shared their work, before their findings were submitted to a physician for final validation. Did they both separately review all records? Did they divide their assigned records between them? Please clarify.\n\nIn your introduction, 2nd paragraph, you review alternative methods for detecting AEs, including incident reporting, CR review, and automated administrative data review (e.g., PSIs). Consider adding “prospective (concurrent) clinical trigger systems,” that track possible real-time clinical responses to AEs then track back to see if an AE actually occurred. Dr. R. Scott Evans at LDS Hospital in Salt Lake City, Utah; and Dr. David Bates at Brigham & Women’s Hospital in Boston, Massachusetts, developed and demonstrated such systems. Such approaches find AEs that never make their way into a traditional clinical record, and may detect far more events.\n\nUnder Methods, please change the heading “Sampling selection” to say something like “Sample selection.” More importantly, the frame within which “10 inpatient CRs” were selected monthly is not clear. Working the total number of charts reviewed backward, you were probably sampling “10 inpatient CRs” each month for each of the 44 Sicilian public hospitals. Please clarify. How was record selection balanced across the types of wards (medicine, surgery, obstetrics, and ICUs) in each hospital?\n\nUnder Results, please add clarity regarding the number of patients that had at least 1 AE during their index hospitalization (975); then break out the number of patients who experienced a single AE versus those who had more than 1 AE during their index hospitalization.\n\nTable 1: Please clarify – I can’t understand what you mean by the phrases “CRs with triggers per inpatient wards” versus “CRs with trigger isolated per wards”; or “CRs with trigger” versus “CRs with trigger isolated”.\n\nConsider moving the first 2 paragraphs of the section entitled “Rates of triggers” from the Discussion section into the Results section.\n\nClarify your text in the first paragraph of your “Rates of triggers” section. For example, it might read:\n“In this study, 37.9% (n=5,574) of all CRs examined had at least 1 positive trigger. Of those, 2,778 CRs had a single positive trigger (49.8% of all CRs with positive triggers) while 2,796 CRs had more than one positive trigger (51.2% of all CRs with positive triggers).\n\nConsider modifying Table 2:\na) Label each of the subsections in Table 2. For example, in your text you cite the Table relative to “general care triggers”. What are “general care triggers”? Presumably you mean the subset of triggers with “C” labels. It would be quite helpful if you put headings on each of the subsections (for C, M, S, P, and I) that labelled the contents of each subsection.\nb) You are (quite appropriately) approaching AE detection as a screening test with 2 main steps. Step 1 involves evaluation of the initial triggers. Step 2 involves following up on positive initial triggers, to see if a validated AE emerges. This is done in the context of a general review by a trained clinical expert, who can (and sometimes does) detect AEs that fall outside of the trigger system.\nIt would be very useful if you separated the total time spent on CR review into (a) initial assessment of the triggers; versus follow-up analysis of the positive triggers.\n\nIt would similarly be very useful if, in the 3rd column of Table 2 (labelled “Times associated with AEs, n (%)” you showed the proportion of positive triggers that yielded a confirmed AE related to/deriving from the initial positive trigger. The Table, as currently constructed, is quite confusing – in 10 instances (C03, C04, C08, C11, C12, C14, M02, M04, I01, I03) the count in 3rd column is larger than the count in 2nd column. Presumably, this is because you are not connecting the trigger to derivative AEs (but I can’t really tell, with any certainty).\n\nThe section entitled “Rates of AEs”, first paragraph, contains the sentence:\n\n“It would appear that the percentage of clinical records with triggers increases with the increase of percentage of CRs examined, compared to patients discharged, while the percentage of CRs with AEs remains more stable.”\n\nI can’t translate quite what that means. Please simplify and clarify. It may relate to the 2 upper lines in Figure 2, which both appear to be increasing over time. However, you supply no statistical analysis to show that trends over time in the 2 lines are statistically associated, and you offer no reasoning as to why such a relationship may have meaning.\n\nTable 3 appears to identify high frequency triggers, then count the number of AEs of any sort that occur in CRs that had a high frequency trigger. It would be much more useful if you tracked each trigger to related or derivative AEs, rather than treating AEs generically.\n\nIn the section labelled “Rates of AEs” you have a bulleted list. The last item in that list says:\n\n“Intensive-care-related triggers have been identified 1,817 times with an AE in 1,924 (i.e., it is very common for triggers to identify more AEs in the same patient)”.\n\nI think I get the gist of what you mean, but (a) this sentence needs more clarity; and (b) the main purpose for identifying AEs is to move toward intervention and prevention (better, safer care). In that circumstance, treating AEs as generic items is not very useful. It is necessary to list them by specific causes. In that framing, associating triggers (which are cause specific) with a total generic AE count is not very informative or useful. You might want to rethink your framing.\n\nSpelling corrections:\nIn the Abstract, Methods subsection, it should say “300,000 inpatient yearly admissions”.\n\nIn the Methods section, it should say “Sample selection,” not “Sampling selection”.\n\nIn the Discussion section, 2nd paragraph, it should say “A critical appraisal of the studies and their results is difficult, as the methodologies use are heterogeneous,”.\n\nIn Figure 1, the label for the blue line should say “Sensitivity (%),” not “Sensibility (%)”.\n\nIn the section labelled “Rates of AEs” there is a bulleted list. The 4th bullet should say “Surgery-related” (not “Surgery-elated”).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4674", "date": "28 May 2019", "name": "vincenzo parrinello", "role": "Author Response", "response": "We appreciate the review by Professor Btrent of our paper, the accuracy of his observations and the relevance of his suggestions. We are honored that he considers our paper “useful because it supplies empiric observation of one such use, across a large system of hospitals and an extended period of time.” We are aware that some of the points require further clarity, which may have been lost as we limited the word count. Comment The Harvard Medical Practice Study (HMPS – cited by the authors) used post-discharge chart review to detect care-associated injuries (adverse events – AEs) that occurred during hospitalization. Under HMPS, trained nurses reviewed a random selection of charts. If those nurses discovered what they judged to be care-associated injuries, they flagged those events in the chart as a potential AE. Charts that contained one or more AEs were forwarded to 2 independent physician reviewers. If both physician reviewers judged, for each AE, that event had occurred, then it was reported as a confirmed AE. Those physicians also judged whether each AE was avoidable, and whether each could be considered negligent (substandard) care. The IHI Global Trigger Tool (IHI GTT) built on the HMPS methodology. It attempted to improve the ability of initial nurse reviewers to detect potential AEs, by providing a set of 51 “review triggers,” falling into 7 major subcategories – explicit initial events that, if detected in the chart, chained to examination of other specific events. Initial assessments of the IHI GTT showed much higher detection rates for AEs than did the original HMPS methodology, which itself far exceeded typical voluntary reporting mechanisms. Response Harvard's medical practice study is a key pillar in patient safety and care research. We agree that the IHI Global trigger tool is an attempt to improve the Harvard Medical Practice Study methodology. In our opinion, the IHI Global Trigger Tool has two significant differences from the Harvard medical practice study. The first is that the IHI Global Trigger Tool is conducted on a random sample of clinical records that are analyzed only if triggers are present. The second is that the IHI Global Trigger Tool \"focuses on only those adverse events related to the active delivery of care (commission) and excludes, as much as possible, issues related to substandard care” (omission). (IHI Global Trigger Tool for Measuring Adverse Events. Second Edition 2009. http://www.ihi.org/resources/Pages/Tools/IHIGlobalTriggerToolforMeasuringAEs.aspx) Comment This report has at least 2 major differences from other work in the field. First, only 6.6% of records reviewed had at least one care-associated adverse event. Other studies have shown much higher AE rates. The authors note this in their text, and suggest it may reflect differences in local environments and chart review methods. Response The percentages of adverse events found in other studies are very different. In a recent systematic review of the literature, the percentage of adverse events is between 7% and 51%. (Hibbert PD, Molloy CJ, Hooper TD, et al .: The application of the Global Trigger Tool: a systematic review. Int J Qual Health Care. 2016; 28 (6): 640-649.) We think that the most important reasons, which can explain these wide differences, could be twofold. The first could be due to the different care settings (medicine, surgery, obstetrics, ICU) and therefore to the different complexity of the patients studied. A risk adjustment system would be needed to evaluate the results homogeneously. In our experience, for example, there are significant differences in the AEs rate between apparently homogeneous wards belonging to different hospitals. We have not included these tables to be brief. Since it seems to us that this is very interesting, this aspect will be the subject of other publications. The second reason could be due to changes in the detection protocol. The survey methods are, in fact, different in the different studies. We, for example, have turned our attention only to the triggers observed during admission and not to the triggers present at the time of admission. Therefore the adverse events found do not include those present at the time of admission. This could justify the differences in EAs observed compared to other studies. Comment Second, most other IHI GTT reviews show adverse drug events (medication-related events - overdoses, drug-drug interactions, and allergic or idiosyncratic reactions) as the dominant category of AEs detected. In this study, they are a distant number 3. Why? Response We do not know why the drug-related AEs observed in our study are infrequent compared to other studies. In our study the most frequently detected AEs are Health care–associated Infection. In addition, Rutberg et al. Report that Health care–associated Infection accounted for 39% of Aes. (Rutberg H, Borgstedt Risberg M, Sjödahl R, et al. Characterization of adverse events detected in a university hospital: a 4-year study using the Global Trigger Tool method. BMJ Open. 2014; 4: e004879.) Von Plessen and Coll report that Infections, pressure ulcers procedures-related and gastrointestinal problems were common. (Von Plessen C, Kodal AM, Anhøj J. Experiences with Global Trigger Tool reviews in five Danish hospitals: an implementation study. BMJ Open. 2012; 2: e001324. Schildmeijer et al report that \"Overall, the level of agreement for detecting AEs is the level of harm for healthcare-related infections, that is, pneumonia, sepsis and urinary tract infection.\" (Schildmeijer K, Nilsson L, Årestedt K, et al. Assessment of adverse events in medical care: lack of consistency between experienced teams using the global trigger tool. BMJ Qual Saf. 2012; 21: 307-314.) Landrigan et al. report that \"Harms that were detected in procedures (186/588)\" (Landrigan CP, Parry GJ, Bones CB, et al. Temporal trends in rates of patient harm resulting from medical care. N Engl J Med. 2010; 363: 2124-2134.) Mortaro et al. report that the most common types of AEs detected were related to surgical procedures (Mortaro A, Moretti F, Pascu D, et al .: Adverse Events Detection Through Global Trigger Tool Methodology: Results From a 5-Year Study in an Italian Hospital and Opportunities to Improve Interrater Reliability. J Patient Saf. 2017.) Comment It appears that the entire IHI GTT program assessed a total of 18,008 records from among 105 hospital wards from June 2015 through June 2018. However, this analysis examines only medicine, surgery, obstetrics, and intensive care wards. Those represented a total 89 wards, with a total of 14,706 records reviewed. This is unclear in both your abstract and your text. It would be very helpful to more clearly explain how you derived the CRs included in your analysis. Response We agree that this may be unclear. From June 2015 to June 2018, 18.008 CRs were examined. In this study, we analyzed 14,706 CRs relating to patients discharged from 89 medicines, surgery, obstetrics and intensive care wards. 16 wards and 3,302 CRs concerned pediatrics wards and emergency department. Comment You mention that you had a total of 199 individuals who participated in 71 3-person review teams. Obviously, some people participated in more than 1 team. It is not clear how the 2 initial reviewers shared their work, before their findings were submitted to a physician for final validation. Did they both separately review all records? Did they divide their assigned records between them? Please clarify. Response The total number of reviewers was 199 divided into 71 teams. In some teams, medical reviewers from another team were the supervisors. The two primary record reviewers should each review all records independently. The third reviewer was always a physician. The physician, who did not review the records, authenticated the consensus of the two primary record reviewers. Comment In your introduction, 2nd paragraph, you review alternative methods for detecting AEs, including incident reporting, CR review, and automated administrative data review (e.g., PSIs). Consider adding “prospective (concurrent) clinical trigger systems,” that track possible real-time clinical responses to AEs then track back to see if an AE actually occurred. Dr. R. Scott Evans at LDS Hospital in Salt Lake City, Utah; and Dr. David Bates at Brigham & Women’s Hospital in Boston, Massachusetts, developed and demonstrated such systems. Such approaches find AEs that never make their way into a traditional clinical record, and may detect far more events. Response This is very interesting. In part, this concept is reported in the conclusions \"The introduction of electronic medical records would allow a quicker process with the automation of the identification of triggers and the possibility of linking together in the identification of adverse events and near misses, especially where there has been more than one trigger \" We will review the work and attempt to explain this concept better. Comment Under Methods, please change the heading “Sampling selection” to say something like “Sample selection.” More importantly, the frame within which “10 inpatient CRs” were selected monthly is not clear. Working the total number of charts reviewed backward, you were probably sampling “10 inpatient CRs” each month for each of the 44 Sicilian public hospitals. Please clarify. How was record selection balanced across the types of wards (medicine, surgery, obstetrics, and ICUs) in each hospital? Response We will change the heading \"Sampling selection\" to \"Sample selection.\" We randomly selected 10 CRs for each department that participated, not for each hospital. The wards that participated were not homogeneously represented in the different hospitals. In some hospitals only the ICU CRs were analyzed. In some hospitals there were no medical, surgical or obstetrics or ICU wards. In others there were more than one medicine, surgery, obstetrics and ICU wards. In addition, hospitals were recruited at different times. The CRs sample is therefore not representative. The aim of our study was not to provide a true representation of the patient population present in hospitals during the observation period. Coherently with as reported with the IHI GTT protocol, the main purpose of applying GTT methodology is to produce a sampling approach that is sufficient for the design of safety work in the hospital. Comment Under Results, please add clarity regarding the number of patients that had at least 1 AE during their index hospitalization (975); then break out the number of patients who experienced a single AE versus those who had more than 1 AE during their index hospitalization. Response We will indicate the number of patients who experienced a single AE versus those who had more than 1 AE during their index hospitalization. Comment Table 1: Please clarify – I can’t understand what you mean by the phrases “CRs with triggers per inpatient wards” versus “CRs with trigger isolated per wards”; or “CRs with trigger” versus “CRs with trigger isolated”. Response We will modify the table. The second line \"CRs with triggers for inpatient wards\" indicates the number and percentage of CRs with one or more triggers. The third line \"CRs with trigger isolated for wards\" indicates the number and percentage of CRs with only triggers. The fourth and fifth lines are repetitions. It it is a printing error. Comment Consider moving the first 2 paragraphs of the section entitled “Rates of triggers” from the Discussion section into the Results section. Response We will move the first 2 paragraphs of the section entitled \"Rates of triggers\" from the Discussion section into the Results section. Comment Clarify your text in the first paragraph of your “Rates of triggers” section. For example, it might read: “In this study, 37.9% (n=5,574) of all CRs examined had at least 1 positive trigger. Of those, 2,778 CRs had a single positive trigger (49.8% of all CRs with positive triggers) while 2,796 CRs had more than one positive trigger (51.2% of all CRs with positive triggers). Response Thank you for this good advice. We will modify accordingly. Comment a) Label each of the subsections in Table 2. For example, in your text you cite the Table relative to “general care triggers”. What are “general care triggers”? Presumably you mean the subset of triggers with “C” labels. It would be quite helpful if you put headings on each of the subsections (for C, M, S, P, and I) that labelled the contents of each subsection. Response We will put headings on each of the subsections (for C, M, S, P, and I) that labelled the contents of each subsection. Comment b) You are (quite appropriately) approaching AE detection as a screening test with 2 main steps. Step 1 involves evaluation of the initial triggers. Step 2 involves following up on positive initial triggers, to see if a validated AE emerges. This is done in the context of a general review by a trained clinical expert, who can (and sometimes does) detect AEs that fall outside of the trigger system. It would be very useful if you separated the total time spent on CR review into (a) initial assessment of the triggers; versus follow-up analysis of the positive triggers. Response We are unclear of the changes required on this point. Comment it would similarly be very useful if, in the 3rd column of Table 2 (labelled “Times associated with AEs, n (%)” you showed the proportion of positive triggers that yielded a confirmed AE related to/deriving from the initial positive trigger. The Table, as currently constructed, is quite confusing – in 10 instances (C03, C04, C08, C11, C12, C14, M02, M04, I01, I03) the count in 3rd column is larger than the count in 2nd column. Presumably, this is because you are not connecting the trigger to derivative AEs (but I can’t really tell, with any certainty). Response In the third column of table 2 we reported the number of times a trigger was found. In the fourth column the number of times in which an adverse event was related to that trigger. The title of the fourth column (Times associated, with AE)does not express this clearly. Therefore in the third column the triggers are indicated, while in the fourth column the adverse events are indicated. We have found some CRswith more than one adverse event. In the third column of table 2 we intended to report that the C03 trigger was found in 279 CRs examined. Instead, in the fourth column of Table 2 we wanted to indicate that in 438 adverse events the C03 trigger was present. Comment The section entitled “Rates of AEs”, first paragraph, contains the sentence: “It would appear that the percentage of clinical records with triggers increases with the increase of percentage of CRs examined, compared to patients discharged, while the percentage of CRs with AEs remains more stable.” I can’t translate quite what that means. Please simplify and clarify. It may relate to the 2 upper lines in Figure 2, which both appear to be increasing over time. However, you supply no statistical analysis to show that trends over time in the 2 lines are statistically associated, and you offer no reasoning as to why such a relationship may have meaning. Response We mean that we did not find a correlation between the number of CRs examined in the period and the number of EAs observed in the same period. We will try to verify if the differences are statistically significant. Comment Table 3 appears to identify high frequency triggers, then count the number of AEs of any sort that occur in CRs that had a high frequency trigger. It would be much more useful if you tracked each trigger to related or derivative AEs, rather than treating AEs generically. Response This would be very interesting to describe, but we do not know if it will be possible to provide this kind of detail due to editorial guidelines. It could be the subject of a new study to be published later. Comment In the section labeled \"Rates of AEs\" you have a bulleted list. The last item in that list says: \"Intensive-care-related triggers have been identified 1,817 times with an AE in 1,924 (i.e., it is very common for triggers to identify more in the same patient)\". I think I get the gist of what you mean, but (a) this sentence needs more clarity; and (b) the main purpose of identifying AEs is to move towards intervention and prevention (better, safer care). In that circumstance, treating AEs as generic items is not very useful. It is necessary to list them by specific causes. In that framing, associating triggers (which are cause specific) with a total generic AE count is not very informative or useful. You might want to rethink your framing. Response This would also be interesting to describe. We have been required to be fit in with te word count and to comply with the editorial guidelines. We will clarify this section" } ] } ]	1	https://f1000research.com/articles/8-263
https://f1000research.com/articles/7-1801/v1	15 Nov 18	{ "type": "Research Article", "title": "Prediction of recombinant Mycobacterium tuberculosis α-crystallin oligomer chaperone activity using polynomial graphs", "authors": [ "Gautam Krishnan", "Utpal Roy", "Gautam Krishnan" ], "abstract": "Background: Mycobacterial α-crystallin (Acr) is a chaperone that prevents misfolding of proteins when Mycobacterium tuberculosis is found in a latent form in the host tissue. Methods: Using insulin as a model substrate and utilizing polynomial graphs, we attempted to predict molecular-level interactions that are a function of the oligomeric state of the recombinant protein. The chaperone activity of the recombinant oligomeric Acr was measured at 60°C with Acr samples obtained before gel filtration chromatography and compared with a gel-filtered sample. Results: The polynomial graphs constructed showed improved molecular coverage of the insulin B chain by the oligomer. The 2nd order coefficient is the one that changes with the oligomeric ratio of Acr and improves chaperone activity. Polynomial analysis suggested that it could be a useful parameter to predict chaperone activity for potential in vitro batches of M. tuberculosis Acr based on the dynamic nature of the association and disassociation of oligomers. Conclusions: The results showed that coverage of insulin B chain improved with higher ratio of 9-mer as compared to lower ratios. This was shown by both simulation plots and actual assay data. The polynomial graphs showed increase in the 2nd order coefficient, thus suggesting the important role of oligomerisation in improved molecular coverage of insulin B chain.", "keywords": [ "Alpha-crystallin", "Chaperone", "Purification", "Recombinant", "Polynomial" ], "content": "Introduction\n\nMycobacterial α-crystallin (Acr) is an important protein in latent tuberculosis (TB). When infected by Mycobacterium tuberculosis, the host survives the infection, yet in 1 out of 3 cases, the bacteria survive in a hypoxic state (Sherman et al., 2001). The alternative to in vivo studies is to clone and express recombinant Acr in Escherichia coli and study its chaperone activity in vitro (Gu et al., 2002; Panda et al., 2017; Yang et al., 1999). Substrates used for past enzymatic studies have included citrate synthase and insulin (Chang et al., 1996; Gu et al., 2002; Mao et al., 2001; Panda et al., 2017). However, there is no precise molecular means to quantify the biological activity of Acr in terms of molecular interactions. Acr is active as an oligomer and though its mechanism of action is known to be in the form of a trimer of trimers (Chang et al., 1996) or in the form of a dodecamer (Kennaway et al., 2005; Panda et al., 2017), yet it is difficult to study the mechanism by which it inhibits the aggregation of substrates, either thermally or by dithiothreitol (DTT) induced with insulin.\n\nIn the present study, we analysed recombinant Acr preparations in terms of chaperone activity against the insulin B chain substrate, and explored the possibility of using the polynomial graphs in predicting chaperone activity. A theoretical and mechanistic representation using the real data has been used to render the numerical predictions of activity based on polynomial graphs.\n\n\nMethods\n\nThe acr gene of M. tuberculosis H37Rv was directionally cloned using the expression vector pET28a as an N-terminal His tag into pET28a using Nde1 and Xho1 restriction enzymes.(Takara). Expression of the recombinant acr was initially done at 50 ml scale, using 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) induction (37°C for 3h). The cell pellets obtained were used for the SDS-PAGE (15% acrylamide) to check for the expression of protein. The rest of the sample was sonicated using 10 mM Tris pH 7.0 / 5% glycerol followed by centrifugation at 20,000 g at 4°C for 30 mins. Aliquots of supernatant and pellet obtained were used for the 15% SDS-PAGE gel to observe the localization of protein in supernatant or pellet.\n\nRecombinant Acr was purified using Nickel-NTA agarose. The cell pellet obtained after 0.5–1.0 mM IPTG induction was lysed by sonication. The sonicate was centrifuged at 20,000g for 30 mins at 4°C and the supernatant obtained was allowed to bind to a 3 ml of Nickel-NTA resin. The protein was eluted with a 3-step gradient of 300 mM, 400 mM and 500 mM imidazole in buffer containing 20 mM Tris pH 7.0, 300 mM NaCl and 5% glycerol.\n\nThe Nickel-NTA eluted fractions containing the Acr were dialysed against 20mM Tris pH 7.0, 100 mM NaCl and 5% glycerol and subjected to gel filtration chromatography on a Sephacryl-200 Hiprep XK 16/60 Column (Pre-packed 120 ml) using the AKTAPurifier system (GE Healthcare Life Sciences). The column was equilibrated using the same buffer and calibration carried out using BIORAD standards, Molecular weight ranging from 670 kDa to 1.5 kDa. SDS –PAGE analysis was carried out with the first 3 lanes loaded with non-gel-filtered samples and the lanes 5–10 with 2 eluted peaks C1 and C2 of different runs with varying amounts of beta mercaptoethanol. A Native-PAGE analysis was carried out for both non-gel-filtered and gel-filtered samples, respectively using 8–16% gradient Tris glycine gel, and bovine serum albumin (Hi-Media; 0.5 mg/ml) as a standard. The oligomer size was estimated using a plot of log molecular weight versus distance migrated and estimated sizes calculated using antilog.\n\nActivity of recombinant M. tuberculosis Acr was assessed at 60°C using three concentrations (83, 118 and 125 μM) of insulin B chain as a substrate, by adding together Acr along with the insulin B chain. Aggregation of the insulin B chain was initiated by addition of 25 mM DTT and the change in the absorbance was monitored at 360 nm for 20 mins in kinetic mode with 1800 time points of 1 second interval. The assay was carried out at 60°C with His-tag-eluted samples at single point three concentrations of 12, 30 and 37.5 μM labelled as non-gel-filtered samples, and also with a His-tag plus gel-filtered sample at a concentration of 12 μM. The assay volume for 12 μM Acr was 400 microlitre while the other 2 assays were done in assay volume of 250 microlitres. Later assays were done at single-point concentrations of 11 µM and 37.5 µM at 60°C (data not shown).\n\nA set of simulation graphs was plotted in MS Excel 97-2003 assuming different ratios of oligomers and the number of molecules of Acr versus the number of molecules of insulin at 118 μM and the percentage of molecules of insulin B chain covered. The assumption was 4 molar concentrations of 5, 10, 20 and 40 μM and 2 different proportions of 9-mer to 12-mer oligomers; 60% of 9-mer and 10% each of 10- to 12-mer and 20% each of 9-mer to 12-mer. This gave an indication about the trends in percentage insulin B chain covered at two different ratios in scenarios, one with higher amount of nonamer and the second a lower amount of nonamer.\n\nThe assay data we obtained was used to calculate the percentage inhibition of insulin aggregation versus percentage of molecules of insulin B chain covered with both monomers and oligomers for non-gel-filtered sample. The assumption made was the molecular size of Acr to be 18 kDa as monomer and further plots were constructed assuming the proportion of 9-mers to 16-mers as obtained from Native-PAGE data. The polynomial plots were used to gain insights into the binding of Acr to the insulin B chain.\n\n\nResults\n\nThe His-tag purification and gel filtration showed greater than 95% purity as seen in the gel analysis along with upper (higher) molecular weight bands (Figure 1a). The protein appeared in the void volume suggested a blend of oligomers which was later confirmed by Native-PAGE analysis.\n\n(a) SDS-PAGE of gel filtration runs 1 and 2. Lanes 1, 2, 3: Load -1X and 2Xβ-mercaptoethanol (β-ME); lane 4: marker showing 14, 20, 22, 25, 35, 43, 50, 56, 66 and 80 kDa; lanes 5-7: run 1 C1 1X, 2X and 3X β-mercaptoethanol; lanes 8-10: run 2 C2 1x, 3x and 2x β-ME. (b) Native-PAGE of His-tag-eluted non-gel-filtered samples and gel-filtered elute. Lane 1, 9: BSA control; lane 3: non-gel filtered; lanes 4, 5, 6: gel-filtered; lane 7, 8: non-gel-filtered. (c) Plot of log molecular weight of the different forms of BSA in Native-PAGE. The different sizes of BSA 66, 132 and 198 kDa were plotted against distance migrated and the log molecular weight was plotted in MS excel and the equation displayed and used to calculate oligomer size of non-gel-filtered and gel-filtered samples.\n\nNative-PAGE analysis showed a proportion of 70% of 9-mer and 10% each of 10-mer to 12-mer in the non-gel-filtered (before the gel-filtration chromatography) samples and 40% of 16-mer and 15% each of 9-mer to 12-mer as analysed by ImageJ software (Figure 1b, c and Table 1).\n\nThe Native-PAGE data was used to calculate the oligomeric sizes and ratios of different bands in non-gel-filtered and gel-filtered samples Oligomer sizes were calculated using bovine serum albumin (BSA) monomers, dimers and trimers as a reference (66, 132 and 198 kDa) and the log of molecular weights plotted versus the distances travelled in centimetre. From this calculation, molecular weight was extrapolated for the different bands seen in non-gel-filtered samples (band 1, 2, 3, 4) and gel-filtered samples (band 1, 2, 3, 4 and 5).\n\nThe acr-pET28a construct showed activity against DTT induced aggregation of insulin. At a concentration of 37 μM of Acr and 125 μM insulin it gave 40% inhibition (Figure 2a). At a concentration of 12 μM Acr and 83 μM insulin it gave 60% inhibition (Figure 2b). A separate assay was carried out using both non-gel-filtered and gel-filtered samples at 12 and 30 μM, respectively and insulin at 118 μM concentration (Figure 2c). There was a difference in activity between both His-tag and gel-filtered samples. The non-gel-filtered samples showed 21.54% inhibition at a concentration of 30 μM, whereas 60% inhibition was achieved by the gel-filtered sample at a concentration of 12 μM Acr.\n\n(a) Insulin was assayed at a concentration of 125 µM with α-crystallin (Acr) at a concentration of 37.5 µM in an assay volume of 400 µl by adding Acr to prevent DTT induced aggregation of B chain. (b) Insulin was assayed at a concentration of 83 µM with Acr added at a concentration of 12 µM in an assay volume of 250 µl. (c) Insulin was assayed at a concentration of 118 µM and using two concentrations of non-gel-filtrered Acr (30 µM) and gel-filtered Acr (12 µM), in an assay volume of 250 µl.\n\nActivity of Acr was plotted in terms of percentage inhibition versus percentage of molecules of insulin B chain and polynomial graphs constructed to check for best fit analysis with R2 greater than 0.95 being the criteria (Figure 3a, b). The graphs showed 2nd order polynomials as the best fit. The coefficient of x2 was higher in the oligomer plot, indicating that oligomerisation decreases the percentage of molecules of the insulin B chain covered. This was performed for the non-gel-filtered samples for both monomer and oligomer, assuming the ratios obtained from Native-PAGE analysis. The simulations showed the proportion of oligomers required to cover a maximum percentage of insulin B chain changed with the proportion of 9-mers with higher proportion of 9-mer covering more of the insulin B chain (Figure 4a, b).\n\n(a) A polynomial plot was used to plot percentage inhibition of non-gel-filtered α-crystallin (Acr) monomers against percentage of of molecules of the insulin B chain covered at 60°C. (b) A polynomial plot was used to plot percentage inhibition of non-gel-filtered Acr oligomer versus the percentage of molecules of insulin B chain covered at 60°C. (c) The percentage inhibition was replotted versus percentage of molecules of insulin B chain at 60°C by calculating the molecules of Acr based on the proportion of oligomers obtained from Native-PAGE analysis at 37°C.\n\n(a) A plot of simulated Acr oligomer assuming 9-mer ratio of 60% and 10% of 10- to 12-mer versus percentage of molecules of insulin B chain covered at 118 µM. (b) A plot of simulated Acr oligomer assuming a 9-mer to 12-mer ratio of 20% versus the percentage of molecules of insulin B chain covered at 118 µM.\n\n\nDiscussion\n\nHis-tag purification attempts of the recombinant Acr-producing construct showed the protein obtained was greater than 95% purity in the fractions eluted with 500 mM imidazole. The chaperone activity varied with two different concentrations of Acr, and the plots of monomer versus oligomer obtained showed that oligomerisation helps more molecular coverage the insulin B chain. This was confirmed by the results obtained by the gel-filtered samples that showed higher inhibition than the non-gel-filtered samples. This could be possibly explained by the higher proportion of 10-mer to 12-mer and 40% of 16-mer, which indicates that a higher proportion of oligomers improves chaperone activity.\n\nThe generation of polynomial graphs plotted showed an increase in the x2 coefficient of the 2nd order graph, which is directly proportional to the amount of oligomers present and can be possibly used to model chaperone activity using insulin as a substrate. These observations suggest that the change in the percentage of molecules of insulin B chain covered is due to the extent of oligomerisation that forms and also varies with the ratios of oligomers directly affecting the activity, suggesting that this is a significant parameter for checking in vitro Acr activity.\n\nPolynomial graphs have been used to analyse activity of proteins involving oligomers (Yeow & Clayton, 2007). Many oligomer proteins function as high-molecular-weight aggregates and polymers, and the mechanism of heat-shock proteins is for that reason, poorly understood (Mattoo, 2014). This approach of using polynomials can shed insights into the molecular mechanism of oligomers binding to substrates.\n\n\nConclusions\n\nUsing polynomial graph analysis, we suggest a predictive tool using percentage inhibition versus insulin B chain covered for in vitro Acr preparations that could also be extrapolated for in vivo substrates of Acr as a logical outcome for future studies.\n\n\nData availability\n\nDataset 1: All raw data for article ‘Prediction of recombinant Mycobacterium tuberculosis α-crystallin oligomer chaperone activity using polynomial graphs’. Raw, uncropped blots are given in Figure 1a, b. DOI: https://doi.org/10.5256/f1000research.16328.d219535 (Krishnan & Roy, 2018).", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nAuthors acknowledge the Director and the Vice Chancellor of BITS Pilani for providing necessary financial assistance in the form of visiting fellowship and institute contingency. We wish to thank Dr. Santanu Datta of Bugworks, G.K.’s co-supervisor, who provided invaluable technical assistance.\n\n\nReferences\n\nChang Z, Primm TP, Jakana J, et al.: Mycobacterium tuberculosis 16-kDa antigen (Hsp16.3) functions as an oligomeric structure in vitro to suppress thermal aggregation. J Biol Chem. 1996; 271(12): 7218–7223. PubMed Abstract \| Publisher Full Text\n\nGu L, Abulimiti A, Li W, et al.: Monodisperse Hsp16.3 nonamer exhibits dynamic dissociation and reassociation, with the nonamer dissociation prerequisite for chaperone-like activity. J Mol Biol. 2002; 319(2): 517–26. PubMed Abstract \| Publisher Full Text\n\nKennaway CK, Benesch JL, Gohlke U, et al.: Dodecameric structure of the small heat shock protein Acr1 from Mycobacterium tuberculosis. J Biol Chem. 2005; 280(39): 33419–33425. PubMed Abstract \| Publisher Full Text\n\nKrishnan G, Roy U: Dataset 1 in: Prediction of Chaperone Activity of Recombinant M.tb. Acr Oligomer Using Polynomial Graphs. F1000Research. 2018. http://www.doi.org/10.5256/f1000research.16328.d219535\n\nMao Q, Ke D, Feng X, et al.: Preheat treatment for Mycobacterium tuberculosis Hsp16.3: correlation between a structural phase change at 60 degrees C and a dramatic increase in chaperone-like activity. Biochem Biophys Res Commun. 2001; 284(4): 942–947. PubMed Abstract \| Publisher Full Text\n\nMattoo RUH: Mechanistic insights into cytosolic molecular chaperones in protein unfolding and disaggregation. Thesis, Originally published at: University of Lausanne Posted at the University of Lausanne Open Archive http://serval.unil.ch Document URN : urn:nbn:ch:serval-BIB_EA49B7E040F21. 2014. Reference Source\n\nPanda AK, Chakraborty A, Nandi SK, et al.: The C-terminal extension of Mycobacterium tuberculosis Hsp16.3 regulates its oligomerization, subunit exchange dynamics and chaperone function. FEBS J. 2017; 284(2): 277–300. PubMed Abstract \| Publisher Full Text\n\nSherman DR, Voskuil M, Schnappinger D, et al.: Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding alpha -crystallin. Proc Natl Acad Sci U S A. 2001; 98(13): 7534–7539. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang H, Huang S, Dai H, et al.: The Mycobacterium tuberculosis small heat shock protein Hsp16.3 exposes hydrophobic surfaces at mild conditions: conformational flexibility and molecular chaperone activity. Protein Sci. Cambridge University Press. Printed in the USA. 1999; 8(1): 174–179. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYeow EK, Clayton AH: Enumeration of oligomerization states of membrane proteins in living cells by homo-FRET spectroscopy and microscopy: theory and application. Biophys J. 2007; 92(9): 3098–3104. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "40682", "date": "07 Jan 2019", "name": "Anjan Purkayastha", "expertise": [ "Reviewer Expertise Infectious disease", "bioinformatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe Acr protein is a chaperone that prevents misfolding of proteins in latent M. tuberculosis. Characterizing the precise molecular mechanism of action of Acr, however, remains a challenge. The authors, Krishan and Roy, have used a novel means of estimating the degree of oligomerization using polynomial graphs. The authors make two key observations: the coverage of the insulin B molecules is influenced by (a)the degree of Acr oligomerization and (b)the ratio of oligomers. This work lays the groundwork for further experiments in understanding the chaperoning activity of Acr at a molecular level.\n\nIs the work 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": "61501", "date": "30 Jun 2020", "name": "Nasreen Zafar Ehtesham", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 titled “Prediction of recombinant Mycobacterium tuberculosis α-crystallin oligomer chaperone activity using polynomial graphs” by Gautam Krishnan and Utpal Roy demonstrated the relationship between various higher-order oligomers of Acr and its relation to the prevention of aggregation of insulin B chain as a function of its chaperone activity, predicted using a mathematical polynomial graph. Earlier reports showed that Acr exists in multiple higher-order oligomers, which is related to their chaperone functions. Acr is a vital chaperone protein expressed majorly in the stationary phase as well as in hypoxic conditions. It is required for the survival of M. tb in macrophages; thus, it is a critical virulence effector. The authors utilize the previously known pieces of information and use of polynomial graphs in the prediction of Acr oligomers and its relation to its chaperone activity. They devised a novel way to predict Acr chaperone activity and its relation to the oligomeric state of the Acr. The manuscript is well written, organized, and presented. Few minor concerns which are mentioned below should be addressed before finalizing the manuscript for indexing.\n\nIntroduction:\nThe last sentence of the first paragraph needs revision. Minor grammatical error is present.\nResults:\n\nAcr of Mycobacterium tuberculosis exists in multiple oligomeric states, as reported earlier, and also mentioned by the authors in the introduction section. As shown in the denaturing gel picture, only prominent SDS resistant dimeric or trimeric forms are visible, other oligomers are present only in minute quantity. Is the dimeric form being the stable and primary oligomeric state of Acr in M. tb? The authors should also label gel pictures indicating Molecular weight standards and various oligomers.\n\nHow the authors calculated the percentage of various oligomers of Acr? Whether they used three gels run with samples purified in different batches or the results given are from a single gel?\n\nThe sentence starting under the heading “Chaperone activity at 60°C” needs revision. The authors should revise the sentence.\n\nIs the work 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": "5680", "date": "15 Jul 2020", "name": "Gautam Krishnan", "role": "Author Response", "response": "The manuscript titled “Prediction of recombinant Mycobacterium tuberculosis α-crystallin oligomer chaperone activity using polynomial graphs” by Gautam Krishnan and Utpal Roy demonstrated the relationship between various higher-order oligomers of Acr and its relation to the prevention of aggregation of insulin B chain as a function of its chaperone activity, predicted using a mathematical polynomial graph. Earlier reports showed that Acr exists in multiple higher-order oligomers, which is related to their chaperone functions. Acr is a vital chaperone protein expressed majorly in the stationary phase as well as in hypoxic conditions. It is required for the survival of M. tb in macrophages; thus, it is a critical virulence effector. The authors utilise the previously known pieces of information and use of polynomial graphs in the prediction of Acr oligomers and its relation to its chaperone activity. They devised a novel way to predict Acr chaperone activity and its relation to the oligomeric state of the Acr. The manuscript is well written, organized, and presented. Few minor concerns which are mentioned below should be addressed before finalizing the manuscript for indexing. Responses from the authors Introduction:The last sentence of the first paragraph needs revision. Minor grammatical error is present.Acr is active as an oligomer and though its mechanism of action is known to be in the form of a trimer of trimers (Chang et al., 1996) or in the form of a dodecamer (Kennaway et al., 2005; Panda et al., 2017), yet it is difficult to study the mechanism by which it inhibits the aggregation of substrates, either thermally or by dithiothreitol (DTT) induced with insulin.Author’s response: Suggested change►Acr is active in oligomeric state, and though it is known to act as a trimer of trimers (Chang et al., 1996) or a dodecamer (Kennaway et al., 2005; Panda et al., 2017), yet it is difficult to study the mechanism by which it inhibits the thermal aggregation or dithiothreitol (DTT)-induced aggregation of insulin.Results: Acr of Mycobacterium tuberculosis exists in multiple oligomeric states, as reported earlier, and also mentioned by the authors in the introduction section. As shown in the denaturing gel picture, only prominent SDS resistant dimeric or trimeric forms are visible, other oligomers are present only in minute quantity. Is the dimeric form being the stable and primary oligomeric state of Acr in M. tb? The authors should also label gel pictures indicating Molecular weight standards and various oligomers.Author’s response: The dimer and trimer forms are of the Bovine serine albumin (BSA)standard in Lane 1 and 9 (Figure 1a). These are of the standard. The gel image has been relabelled. The predominant form of M.tb Acr is nonamer to 12-mers which could be an aggregation of trimers or dimers. Chang et al, 1996 showed that the repeating unit is a trimer and a trimer of trimers forms a nonamer. We need to do more experimentation to verify the same.The re-labelling of the images has been done as suggested by the Honorable reviewer. As can be seen in Figure lanes 3,7 and 8 there is a proportion of 9 to 12 mers in the non-gel filtered samples as compared to higher proportion of 12 mers in lanes 4,5 and 6.How the authors calculated the percentage of various oligomers of Acr? Whether they used three gels run with samples purified in different batches or the results given are from a single gel?Author’s Response:The percentage of various oligomers was calculated using software called Image J. This was accomplished by measuring the intensity of each band calculating the total intensity and dividing the intensity of each band by the total intensity to obtain the proportion. The molecular weight of oligomers was obtained by comparing with the standard Bovine serum albumin standards. The results are from a single gel. All batches were analyzed similarly and showed a range of oligomers concentrations which could explain the relation between higher oligomer ratio and activity.The sentence starting under the heading “Chaperone activity at 60°C” needs revision. The authors should revise the sentence.Author’s Response:According to the suggestion heading has been changed to ‘Chaperone activity of Recombinant Acr at higher temperature’" } ] } ]	1	https://f1000research.com/articles/7-1801
https://f1000research.com/articles/9-707/v1	15 Jul 20	{ "type": "Opinion Article", "title": "Public research funding and pharmaceutical prices: do Americans pay twice for drugs?", "authors": [ "Rena M. Conti", "Frank S. David", "Rena M. Conti" ], "abstract": "In the debate over prescription drug pricing, some pharmaceutical industry critics claim that U.S. taxpayers pay twice for costly therapies, because publicly supported research is a major contributor to drug discovery and American taxpayers are inadequately rewarded for their research investment due to high drug prices. In fact, the empirical evidence supporting these claims is weak, and the pay twice argument distracts from important efforts to ensure that impactful new drugs continue to be developed and made widely available to patients who need them.", "keywords": [ "Pharmaceutical", "drug", "price", "access", "research", "development", "funding", "patents" ], "content": "Introduction\n\nDo pharmaceutical companies unfairly bilk American patients when they charge exorbitant prices for drugs developed based on publicly funded research? In a hearing of the House Committee on Oversight and Reform in January, 2019, U.S. Representative Alexandria Ocasio-Cortez of New York argued that “the public is acting as early investor, putting tons of money into the development of drugs that then become privatized, and then they receive no return on the investment that they have made” (see video recording from Twitter).\n\nEmbedded in this “pay twice” argument are three key questions. First, to what extent is public funding responsible for the invention of most new drugs? Second, are U.S. taxpayers inadequately rewarded for their contribution to new drug development? And third, how are these claims salient to the national debate over drug prices?\n\nIn this piece, we summarize the available evidence regarding each of these questions as follows: First, although public funds support a significant amount of important biomedical research, the vast majority of the credit for translating this research into new therapies is due to private companies. Second, the contention that taxpayers receive “no return” on their research investment is incorrect, as it fails to account for the massive health and wealth benefits that Americans receive from new drugs. And finally, the pay twice claim distracts attention from far more impactful and feasible efforts to balance innovation and access across the entire pharmaceutical sector.\n\n\nAre new drugs invented by publicly funded researchers?\n\nThere is little debate that public funding of basic science is a critical enabler of drug development1,2. The National Institutes of Health (NIH) is the world’s largest government funder of biomedical research, and makes financial and practical contributions to all stages of it, including pre-clinical scientific investigations, translational medicine, and clinical trials. Detailed case studies reveal that public support has played at least some role in virtually all of the 26 most clinically and commercially significant drugs and drug classes approved over the past several decades3. And several important medicines were solely invented by academic researchers, including the lung cancer therapy pemetrexed, the vitamin D analog doxercalciferol, the inhaled pulmonary vasodilator nitrous oxide, and the vaccine for Haemophilus influenzae type b4.\n\nBut in a large majority of cases, the public sector’s contribution to new drugs has been in the form of early scientific findings, unrelated to current or potential applications. The public sector supported key basic research for 19 of the 26 “transformative” drugs and drug classes cited above, contributed to the actual discovery of a new therapy in just 11, and could claim sole discovery credit in only four cases5. More broadly, although NIH funding supported at least one publication related to each of the 210 new medicines approved by the Food and Drug Administration (FDA) from 2010 to 2016, over 90 percent of those papers were related to the underlying drug target, not the actual therapy itself6.\n\nComprehensive reviews of the genesis of approved drugs confirm that while publicly funded science often characterizes important pathologic processes and identifies potential drug targets, the private sector is the main inventor of most new therapies. A recent study found that for only 25 percent of drugs approved from 2008 to 2017 was there any documented contribution, of any magnitude, to a drug’s initial discovery, synthesis, or key intellectual property by a public sector research institution or academic “spin-off” company7. This finding corroborated a review of approvals from 1998 to 2007, which found that publicly funded research helped either identify the chemical structure of the final compound or its direct antecedents or demonstrated therapeutic proof-of-concept for the target for only about a third of new drugs8. If one uses a more stringent definition of “contribution” based solely on intellectual property, then taxpayers’ role in drug discovery is even smaller; less than 15 percent of new medicines are covered by even a single patent that was either directly issued to a public entity or contains a “government interest statement” acknowledging public funding7,9,10.\n\nGovernment funding makes enormous contributions to medicine by generating novel insights into biology and disease. But accumulated evidence demonstrates that in the majority of cases, it is the private sector, not academia, which translates those insights into new therapeutics.\n\n\nAre U.S. taxpayers inadequately rewarded for their investment in drug development?\n\nA key component of the pay twice argument is that Americans receive an insufficient return from the funds they allot to biomedical research that enables new drug development. But although it is technically true that direct returns to the NIH from licensing royalties comprise a miniscule fraction of the agency’s budget, this strictly transactional assessment ignores the health and wealth benefits that accrue to taxpayers from publicly funded science.\n\nIn fact, the main return on investment American taxpayers expect from supporting biomedical research is in the form of direct benefits to morbidity and mortality – which have largely been realized. Therapies enabled by publicly funded science have extended and improved human lives, and enabled patients to avoid hospitalizations and other costly interventions that yield worse outcomes11–13. In specific therapeutic areas, like hypertension14, mental illness15, some cancers16, HIV17, and routine childhood vaccinations18, biomedical research has generated enormous surplus economic value for the American public, far in excess of the sum of all public and private investments in research and development19. These savings increase further when exclusivity ends, generics enter the market, and low-priced therapies become available to users of both the branded agent and other expensive medicines in the same class20. Many new medicines also generate other valuable health and welfare benefits that are difficult to quantify, such as improving employment and social engagement for both patients and caregivers21.\n\nPublic research and development investments have also been a significant growth driver for the U.S. economy and a wealth creator for taxpayers. This funding has yielded millions of relatively well-paid jobs and billions of dollars of taxes paid into the coffers of local communities, states, and the federal government22, although these benefits take a long time to accrue and are often unevenly distributed across geographies.\n\nAn important caveat to these studies and conclusions is that just because publicly funded biomedical research yields large returns to taxpayers, does not mean that the current system for realizing those benefits is optimal. As discussed above, about 15 percent of new drugs are based on at least one patent that relied on public funding. In almost all cases, those patents were licensed to pharmaceutical firms under the Bayh-Dole Act, which was passed in 1980 to encourage the commercialization of taxpayer-funded research that might otherwise lie dormant23. Since its passage, detractors have argued that academic patenting insufficiently rewards U.S. taxpayers for their contributions, and imposes costs that reduce its net social benefit24. But even in light of possible opportunities to improve Bayh-Dole to create even more social value, the pharmaceutical industry is one of the clearest examples where exclusive intellectual property rights are critical to convert taxpayer-funded research into useful new products, because of the high cost and risk of drug development25.\n\nCalculating the net returns from publicly funded science is complicated, and it is unlikely that economists will ever explicitly quantify them in a way that satisfies all stakeholders. For this reason, it is impossible to determine objectively whether or not the extent of public support for drug development is appropriately accounted for in their prices. But this challenge notwithstanding, it is empirically false to argue that Americans “receive no return on the investment that they have made” in biomedical research.\n\n\nHow is the “pay twice” claim relevant to the debate over high drug prices?\n\nRecent proposals to limit drug prices are motivated by the worthy goal of ensuring that clinically valuable new drugs are not only developed, but also maximally available to the patients who need them26. The pay twice critique has played an increasingly prominent role in justifying these legislative and administrative remedies, fueled by expensive medicines that owe at least some indisputable scientific debt to public research.\n\nFrom a rhetorical standpoint, the pay twice argument certainly brings attention to the challenge of drug access and affordability. But practically speaking, prior experience suggests it would be difficult to link drug prices to the receipt of public support for basic biomedical research. NIH established a “reasonable pricing clause” in 1989 for products developed through some collaborative public-private research grants, which authorized the government to “require … reasonable evidence” of “a reasonable relationship between the pricing of a licensed product, the public investment in that product, and the health and safety needs of the public.” But the agency eliminated this provision in 1995, amidst concerns about how to define “reasonable pricing,” enforce restrictions and penalties, and mitigate potential negative effects on innovation27. These concerns remain relevant today, in light of the persistent challenges outlined above in quantifying these factors, and would likely preclude adoption of a similar policy, especially if it were intended to apply to an even wider set of therapies.\n\nSimilarly, the feasibility of limiting drug prices via existing “march-in” rights is also limited. Bayh-Dole allows the government to obtain a nonexclusive, royalty-free license to patents developed with public funds, for its own use or that of a third party, if the patent holder fails to adequately commercialize the invention. The NIH has denied all of the march-in petitions it has received to date, maintaining that high prices per se are insufficient rationale to claim inadequate commercialization. (A petition related to Exondys 51, a therapy for Duchenne muscular dystrophy, is still under review.) But even if this obstacle were surmounted, the practical impact of march-in rights would be limited, because almost all drugs covered by Bayh-Dole patents are also covered by additional privately held patents, to which the government has no claim28.\n\nBeyond the practical challenge of how to operationalize a link between a drug’s price and the extent to which public funds contributed to its development lies a more fundamental question: why should it matter? If one’s primary concern is that corporate profiteering limits affordable and equitable access to medicines with proven clinical benefits, then the pay twice argument undercuts the potential impact of broader proposals to improve access to all drugs, regardless of their provenance. Recent suggestions discussed by lawmakers and others include revoking monopoly pricing power once a certain profit threshold is exceeded; “delinking” patents from innovation and rewarding innovators instead with prizes; awarding the government more direct control over drug pricing via international reference price or cost-effectiveness benchmarks; and expanding government’s use of so-called “Section 1498” to use or manufacture any patented product29. In parallel, proposals to eliminate out-of-pocket costs and ensure the availability of cheap generics after patent expiry could also improve access to drugs by reducing patient-borne expenses30. These ideas all entail significant tradeoffs related to innovation that are incompletely understood, and reasonable stakeholders can disagree about how to weigh these tradeoffs given this inherent uncertainty31. But importantly, they share a common focus on improving access to all clinically important therapies, irrespective of their origin, while ensuring that new drugs continue to be developed.\n\nIt is superficially attractive to argue that Americans are entitled to pay a lower price for a new drug that was substantially enabled by taxpayer-funded research. But the implication of that claim – that there is no such entitlement to affordability, or far less of one, for a drug mostly developed by a for-profit company – runs counter to the overarching goal of ensuring that all Americans have equitable access to beneficial therapies. Proposals to control a therapy’s price based on the degree to which public funds contributed to its development are not just unfeasible to implement, but also a distraction from more far-reaching efforts to improve the affordability of all medicines. Attention should instead be focused on developing practical solutions that ensure that clinically valuable new drugs continue to be developed and are accessible by all patients in need.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "Acknowledgements\n\nWe thank Amitabh Chandra, Yevgeniy Feyman, Rachel Sachs, and Rebecca Wolitz for helpful comments on earlier drafts of this manuscript, and Gabriela Gracia for expert research assistance on manuscript preparation. We also thank the anonymous reviewers at Annals of Internal Medicine, whose helpful suggestions on an earlier version of this manuscript led us to make substantial improvements.\n\n\nReferences\n\nFlier JS: Academia and industry: allocating credit for discovery and development of new therapies. J Clin Invest. 2019; 129(6): 2172–2174. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSpector JM, Harrison RS, Fishman MC: Fundamental science behind today’s important medicines. Sci Transl Med. 2018; 10(438): eaaq1787. PubMed Abstract \| Publisher Full Text\n\nKesselheim AS, Tan YT, Avorn J: The roles of academia, rare diseases, and repurposing in the development of the most transformative drugs. Health Aff (Millwood). 2015; 34(2): 286–293. PubMed Abstract \| Publisher Full Text\n\nSchneerson R, Porter A, Smith DH: Prevention of systemic infections, especially meningitis, caused by Haemophilus influenzae type b: impact on public health and implications for other polysaccharide-based vaccines. JAMA. 1996; 276(14): 1181–1185. PubMed Abstract \| Publisher Full Text\n\nChakravarthy R, Cotter K, DiMasi J, et al.: Public-and private-sector contributions to the research and development of the most transformational drugs in the past 25 years: From theory to therapy. Ther Innov Regul Sci. 2016; 50(6): 759–768. PubMed Abstract \| Publisher Full Text\n\nCleary EG, Beierlein JM, Khanuja NS, et al.: Contribution of NIH funding to new drug approvals 2010 – 2016. Proc Natl Acad Sci U S A. 2018; 115(10): 2329–2334. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNayak RK, Avorn J, Kesselheim AS: Public sector financial support for late stage discovery of new drugs in the United States: cohort study. BMJ. 2019; 367: l5766. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKneller R: The importance of new companies for drug discovery: origins of a decade of new drugs. Nat Rev Drug Discov. 2010; 9(11): 867–882. PubMed Abstract \| Publisher Full Text\n\nSampat BN, Lichtenberg FR: What are the respective roles of the public and private sectors in pharmaceutical innovation? Health Aff (Millwood). 2011; 30(2): 332–339. PubMed Abstract \| Publisher Full Text\n\nStevens AJ, Jensen JJ, Wyller K, et al.: The role of public-sector research in the discovery of drugs and vaccines. N Engl J Med. 2011; 364(6): 535–541. PubMed Abstract \| Publisher Full Text\n\nLichtenberg FR: Sources of US longevity increase, 1960–2001. Quart Rev Econ Finance. 2004; 44(3): 369–389. Publisher Full Text\n\nLichtenberg FR: Benefits and costs of newer drugs: an update. Manag Dec Econ. 2007; 28(4–5): 485–490. Publisher Full Text\n\nZhang Y, Soumerai SB: Do newer prescription drugs pay for themselves? A reassessment of the evidence. Health Aff (Millwood). 2007; 26(3): 880–886. PubMed Abstract \| Publisher Full Text\n\nCutler DM: The lifetime costs and benefits of medical technology. J Health Econ. 2007; 26(6): 1081–100. PubMed Abstract \| Publisher Full Text\n\nFrank RG, Berndt ER, Busch AB, et al.: Quality-constant “prices” for the ongoing treatment of schizophrenia: an exploratory study. Q Rev Econ Finance. 2004; 44(3): 390–409. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSun EC, Jena AB, Lakdawalla DN, et al.: An economic evaluation of the war on cancer. J Health Econ. 2010; 29(3): 333–346. PubMed Abstract \| Publisher Full Text\n\nPhilipson TJ, Jena AB: Who benefits from new medical technologies? Estimates of consumer and producer surpluses for HIV/AIDS drugs. Forum for Health Economics & Policy. 2006; 9(2): 1–33. Publisher Full Text\n\nPhilipson TJ, Snider JT, Chit A, et al.: The social value of childhood vaccination in the United States. Am J Manag Care. 2017; 23(1): 41–47. PubMed Abstract\n\nLane J, Bertuzzi S: Measuring the results of science investments. Science. 2011; 331(6018): 678–680. Publisher Full Text\n\nPadula WV, Larson RA, Dusetzina SB, et al.: Cost-effectiveness of tyrosine kinase inhibitor treatment strategies for chronic myeloid leukemia in chronic phase after generic entry of imatinib in the United States. J Natl Cancer Inst. 2016; 108(7): djw003. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNeumann PJ, Sanders GD, Russell LB, et al.: Cost-effectiveness in health and medicine. (Oxford University Press). 2016. Reference Source\n\nGruber J, Johnson S: Jump-starting America: How breakthrough science can revive economic growth and the American dream. (Public Affairs). 2019. Reference Source\n\nSampat BN: Patenting and US academic research in the 20th century: The world before and after Bayh-Dole. Research Policy. 2006; 35(6): 772–789. Publisher Full Text\n\nMazzucato M: The entrepreneurial state: Debunking public vs. private sector myths. (Anthem Press). 2013. Reference Source\n\nOuellette LL, Weires R: University Patenting: Is Private Law Serving Public Values? Michigan State Law Review. forthcoming; Stanford Law and Economics, Olin Working Paper No. 2020; 537. Reference Source\n\nBach PB: New math on drug cost-effectiveness. N Engl J Med. 2015; 373(19): 1797–1799. PubMed Abstract \| Publisher Full Text\n\nWolitz RE: The pay-twice critique, government funding, and reasonable pricing clauses: Georgia State University, College of Law Journal of Legal Medicine Symposium. J Legal Med. 2019; 39: 177–211.\n\nEngelberg AB, Kesselheim AS: Use the Bayh-Dole Act to lower drug prices for government healthcare programs. Nat Med. 2016; 22(6): 576. PubMed Abstract \| Publisher Full Text\n\nBrennan H, Kapczynski A, Monahan CH, et al.: A prescription for excessive drug pricing: leveraging government patent use for health. Yale J Law & Tech. 2016; 18: 275. Reference Source\n\nKolchinsky P: The Great American Drug Deal. (Evelexa Press). 2020.\n\nBagley N, Berger M, Chandra A, et al.: The Orphan Drug Act at 30. In: Innovation Policy and the Economy,. J. Lerner and S. Stern, eds. (University of Chicago Press). 2018." }	[ { "id": "73012", "date": "26 Oct 2020", "name": "Fred D. Ledley", "expertise": [ "Reviewer Expertise Characterizing systems for translational science including science", "public and private sector investments", "measures of public value creation", "and public policy." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well written and referenced article and a significant contribution to the discussion about the relationship between public sector funding for biomedical research and drug prices by two accomplished scholars. We believe this is a meaningful contribution to the literature and should be published.\nWe have several comments and suggestions for the authors and, since this is an open review, we offer these in the context of a more dialectical discussion than we would in a conventional review.\nAs the author’s correctly note, there are two largely unrelated, issues in the “playing twice” policy debates:\nIs the public actually paying for drug development while the company receives all of the profits based on the premise that they developed the drug?\n\nAre taxpayers adequately rewarded for the contribution to new drug development? A corollary to this is the question of the role of Bayh-Dole in providing the public sector with a return.\nWe will address these arguments separately.\nFirst, we would note that quote from Representative Ocasio-Cortez’ relates specifically to the second question, not the question of “paying twice”, and recommend that the author clarify this point.\nWhile the authors are absolutely correct in stating that “the vast majority of the credit for translating this research into new therapies is due to private companies,” we would point out that the framing of this question as relating solely to “translation” unfairly narrows the question. The problem is that, rhetorically framing the question as one that relates specifically to “translation’ or “development,” when the public sector contribution focuses primarily on basic biomedical research, makes their argument essentially a tautology.\nAs the authors note, our research (referenced in the manuscript), demonstrates that >$100 billion in NIH funding was related to the drugs (or their targets) approved 2010-2016 www.pnas.org/content/115/10/2329, with >90% of this funding focused on basic research. We call the authors attention to a recent follow-on study, which shows that >$200 billion in NIH funding was related to the 356 drugs approved for the full decade 2010-2019. www.ineteconomics.org/uploads/papers/WP_133-Cleary-et-al-Govt-innovation.pdf, again, with the large majority focused on basic science. If one assumes that industry spends an average of $1.5 billion in developing each drug, the amount estimated by DiMasi et al,1 pubmed.ncbi.nlm.nih.gov/26928437/ without considering cost of capital, (there is no cost of capital correction in calculating NIH spending), these data argue that the public sector contribution that enables drug discovery and development is of the same order of magnitude as the contribution of the private sector in the penultimate stages of translation. If one considers the range of drug development costs described by Wouters et al jamanetwork.com/journals/jama/fullarticle/27623112, or the substantially lower estimates promulgated by some critics, the contributions of the public and private sectors may be very similar.\nThe point is that BOTH the public sector investment in the enabling science AND the private sector investments in translation are required for drugs to reach the market, and that the overall investment needed to translate a scientific insight into an approved product is much greater than generally appreciated. In this context, public DOES contribute a significant fraction of the overall cost of developing a drug, and DOES pay for the drug itself. Taking this value chain one step further, inasmuch as the revenues from sale of the drug provides the revenue for the large majority of industrial R&D, taxpayers are paying the FULL cost of both the basic research and the applied/translational research and development. Despite this, the public is ALSO paying for the profits of biopharmaceutical companies (https://jamanetwork.com/journals/jama/fullarticle/2762308), and the price of drugs is, in fact, titrated to the desired profit rather than the cost of bringing drugs to market.\n\nIt is important to recognize that capital investments by shareholders contribute only a small fraction of the costs of research and development. The large pharmaceutical companies, which are primarily responsible for the costs of most drug development, distribute substantially more capital to shareholders in the form of dividends and stock buybacks than they raise in the form of stock sales www.ineteconomics.org/perspectives/blog/financialization-us-pharma-industry).\nShareholders play a more significant role in funding R&D expenditures of emerging biotechnology companies. While capital investments in biotechnology companies have increased dramatically since 2015, annual investments in biotechnology companies are historically a small fraction of total R&D spending and not greater than public sector support through the NIH.\nWe have several final suggestions for the authors (i) While we agree with the authors that taxpayers are not technically “paying twice,” we encourage the authors to acknowledge the limited frame of this question. (ii) We encourage the authors to recognize that the public sector is paying the large majority of the costs for R&D through taxes and drug prices, and ALSO subsidizing the distribution of cash to shareholders, who speculate in the success or failure of biopharmaceutical companies, but make little contribution to the development of new drugs.\nSecond, the argument that the public should receive a return on its investment in basic science, which was the basis of Representative Ocasio-Cortez’ question, arises from the work of Dr. Marianna Mazzucato (Dr. Mazzucato has consulted with Representative Ocasio-Cortez). As the authors reference, these arguments are articulated in her book The Entrepreneurial State (referenced in the article), and numerous articles in the academic literature. (e.g. hbr.org/2016/10/an-entrepreneurial-society-needs-an-entrepreneurial-state) Her argument is that government serves as the “investor of first resort” in innovation, investing even before angel or venture investors, who expect high returns on their “risk investments.” Inasmuch as taxpayers are making even earlier investments with implicitly higher risk, Mazzucato argues that the public is similarly entitled to a proportionate return on investment. These arguments are increasingly important in public policy discussions and warrant further elaboration in the article. While the authors are technically correct in calling out the statement that taxpayers “receive no return” on investment as untrue, we would encourage them to refrain from rhetorically limiting the question to the issue of “NO” return.\nThe authors correctly quote an extensive literature demonstrating the health value of effective medicines. We would note, however, that much of this work (particularly the series of landmark papers by Lichtenberg), largely reflect advances made in preventing and treating cardiovascular and infectious diseases, and there is increasing concern about the health value of many recently approved drugs, particularly those approved through expedited pathways. The authors also correctly note that job creation, taxes, and economic growth arising from pharmaceutical innovation constitute a public good and an indirect return on taxpayer investments. We would, however, encourage the authors to use more modest language than “…enormous surplus economic value…” in describing these benefits. We would also note that reference #19 does not appear to be relevant to this statement.\nWe would, however, encourage the authors to recognize that these arguments are undercut by social inequities. The benefits of new drugs are not available to those without adequate insurance/government coverage due to their high price; the jobs created by the biopharmaceutical industry are disproportionately available in selected geographic areas and to individuals with access to higher education; companies go to extraordinary lengths to minimize the taxes they pay to the US government; and the profits generated by high drug prices are often distributed to shareholders who are disproportionately in the highest socioeconomic classes and who also go to great lengths to minimize their taxes. In this context, while the returns may not be as high as anticipated by policy makers due to tax avoidance strategies, and the aggregate or average benefits may not reflect the returns to most taxpayers.\nThe authors should also recognize that the Bayh Dole Act captures a very small fraction of the value created by taxpayer investments in basic science and that the direct returns to the public sector are very limited. By design, Bayh-Dole is relevant only to “subject inventions” and the licenses that provide returns to nonprofit, academic organizations are predicated on patentable subject matter. The problem is that the legal definitions of an “invention” and patentable subject matter require that the inventor establish utility and the ability to reduce their advances to practice. In contrast, the primary focus of taxpayer-funded research is on basic science, which does not require recognized utility, though it may be “use inspired” (Stokes, Pasteur's Quadrant: Basic Science and Technological Innovation, Brookings, 1997) and is, by definition, not focused on enabling reduction to practice. Thus, the vast majority of government funded biomedical research is not covered by the Bayh-Dole Act and there is no formal mechanism for the public sector to receive a direct return on investment.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [] }, { "id": "73200", "date": "04 Nov 2020", "name": "Jacob S. Sherkow", "expertise": [ "Reviewer Expertise intellectual property", "patents", "biotechnology", "bioethics", "regulation" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 succinctly and clearly explain why the \"pay twice\" critique regarding drug pricing in the U.S. is misguided using available evidence drawn from the literature. This is important because, as the authors note, critiques regarding \"excessive\" pricing \"run[ ] counter to the overarching goal of ensuring that all Americans have equitable access to beneficial therapies.\" An excellent summary of a thorny—and unfortunately, persistent—topic in the literature.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-707
https://f1000research.com/articles/9-702/v1	14 Jul 20	{ "type": "Opinion Article", "title": "Misjudging early embryo mortality in natural human reproduction", "authors": [ "Gavin E. Jarvis" ], "abstract": "In 2002, in a judgment relating to the use of the morning-after pill, Mr Justice Munby held that pregnancy begins with the implantation of an embryo into the uterus of a woman. The case involved a large body of expert witness evidence including medical and physiological details of human reproduction. Munby J. emphasised one particular aspect of this evidence: namely, the developmental failure rate of human embryos after fertilisation. Under natural conditions, embryo loss is approximately 10-40% before implantation, and total loss from fertilisation to birth is 40-60% (Jarvis, 2016). By contrast, and based on expert witness testimony, Munby J. stated that not much more than 25% of successfully fertilised eggs reach the implantation stage, and that fewer than 15% of fertilised eggs result in a birth, figures that do not accurately represent scientific knowledge regarding human embryo mortality and pregnancy loss under natural conditions. Rather, these figures were derived from experimental laboratory data and clinical outcomes from in vitro fertilisation treatment. Testimony provided by other expert witnesses directly contradicted these specific numerical claims. In emphasising these figures, Munby J. gave the impression that human embryo mortality is substantially higher than available scientific evidence indicated. In this critique, all the scientific expert witness evidence is presented and reviewed, and an explanation provided for why the emphasised figures are wrong. Whether there are implications of Munby J.’s scientific misjudgment on the legal outcome is for others to consider.", "keywords": [ "Mr Justice Munby", "Smeaton", "morning-after pill", "embryo mortality", "early pregnancy loss" ], "content": "Introduction\n\nIn 2002, a judicial review was considered by the Honourable Mr Justice Munby of the Queen’s Bench Division (Administrative Court) to determine whether the supply of Levonelle (commonly referred to as a morning-after pill (MAP)) by pharmacists amounted, in principle, to a criminal act under section 58 and/or section 59 of the Offences against the Person Act 18611,a. Counsel for the claimant argued that the MAP could act by preventing the implantation of a fertilised egg in the uterus and that its use for this purpose therefore constituted an act intended to bring about a miscarriage. Counsel argued that the supply and use of the MAP for this purpose ought therefore to be regulated in the same way as surgically- or medically-induced abortion, as required by the Abortion Act 1967. A judgment in favour of the claimant would have had a significant social impact on the supply of the MAP.\n\nThe Claimant was John Smeaton, on behalf of the Society for the Protection of Unborn Children (SPUC). The Defendant was The Secretary of State for Health, and the two Interested Parties were Schering Health Care Ltd and the Family Planning Association.\n\nMunby J. held that “the prescription, supply, administration or use of the morning-after pill does not – indeed cannot – involve the commission of any offence under either section 58 or section 59 of the 1861 Act”b.\n\nThe case included evidence from scientific expert witnesses and in his judgment, which Munby J. conceded was “necessarily very long”c, he described and scrutinised aspects of this evidence in detail.\n\nThe primary purpose of this article is to shed light upon one aspect of the scientific expert witness evidence, uncritically accepted and deliberately emphasised by Munby J., which was inaccurate and glaringly inconsistent: namely, the extent of natural embryo mortality in humans in the first week after fertilisation. The nature of the error, the inconsistencies in the expert evidence, and their sources are reviewed and explained.\n\nWhether any legal implications arise from the inconsistent expert witness testimony and judicial error is for others to consider.\n\n\nExpert witness statements\n\nCopies of original expert witness statements used in this critique were made available freely on request from the archives of the Claimant. Civil Procedure Rules concerning the use of witness statements for other purposes indicate that where a witness statement has been put in evidence at a hearing held in public, its use is not restricted to the purpose of the proceedings in which it is servedd. Regarding subsequent use of disclosed documents: they may be used only for the purpose of the proceedings in which they are disclosed, except where they have been read to or by the court, or referred to, at a hearing that has been held in publice. The substantive hearing was held in public and lasted three days, starting on 12th February 2002f. All witness statements referred to in this article were read and referred to by Munby J. No court order has been made restricting or prohibiting the use of these expert witness statements. Finally, it is in keeping with principles of open justice (and academic enquiry) that evidence placed before courts be available for public scrutiny, as confirmed in recent case law2.\n\nA full transcript (redacted of personal information) of all these statements is available on request from the author. In producing the transcript from original copies of court papers, every effort has been made to reproduce the content as accurately as possible. Errors and idiosyncrasies in spelling, grammar and style have been retained. Any errors of transcription are entirely the responsibility of this author, and will be corrected on notification. Where consent to publish has been obtained from the witnesses, full transcripts are directly available in the Underlying data3 as described in the Data availability section. Where consent to publish has not been obtained, only those passages quoted by Munby J. or directly referenced in this article are shown, the remainder being redacted. Transcripts are intended to enable readers to draw their own conclusions about the contents of the judgment and this article.\n\nThe scientific and/or medical expert witness statements were provided to the court by eight “very eminent medical experts”g as summarised in Table 1. Munby J. commended submissions from all parties, both written and oral, as being “uniformly of the very highest quality”h. However, although Munby J. stated that “they were all agreed as to the basic physiology”i, close reading indicates that, with regard to the extent of embryo mortality, this was not the case.\n\nWord counts for the whole documents and for the sections directly related to embryo loss were obtained from transcripts of the witness statements made into Microsoft Word, and include footnotes. Values are rounded to the nearest 10 words. For the whole published article (PB/2), a sampling strategy was employed to estimate the body text word count and rounded to 100 words. Transcripts of the witness statements are in the Underlying data. * The content of Exhibit PNL1 is available online here: https://www.pharmaceutical-journal.com/learning/learning-article/guidance-on-pharmacy-supply-of-ehc/20003892.article.\n\n\nThe incidence of natural human embryo mortality\n\nBefore proceeding, it will be helpful to summarise the key biological stages in normal early human reproduction: coitus, ovulation, fertilisation, embryo development and implantation. Munby J. does a commendable job of synthesising the biological evidence in paragraph 126 of his judgment. Coitus introduces sperm into the lower female reproductive tract and ovulation releases an egg into the upper female reproductive tract. Fertilisation occurs in the fallopian tube when sperm and egg meet and one sperm penetrates the egg: “This can be described as Time 0”j. The fertilised egg develops and becomes a blastocyst after 5–6 days. Around day 7, the blastocyst begins to implant into the lining of the uterus. During implantation, the embryo produces human chorionic gonadotrophin (hCG), detection of which “represents the first reliable opportunity to identify the existence of an embryo”k. Approximately 2 weeks after fertilisation, a woman will miss her menstrual period, the first clear external indication of the presence of a developing embryo.\n\nComplex biologic processes do not work perfectly all of the time, including human reproduction. A recent re-analysis has concluded that pre-implantation embryo loss is approximately 10–40% and that total loss from fertilisation to birth is approximately 40–60%4. In addition, a review of scientific data that contribute to quantitative claims regarding natural pregnancy loss provides a detailed background against which claims made by the expert witnesses regarding the incidence of natural human embryo mortality may be evaluated5. Making sense of these numerical estimates is not easy. To aid understanding, Figure 1 summarises conclusions from these articles alongside the claims of the expert witnesses and numerical estimates from an influential and valuable analysis by Henri Leridon6. Details of how the figure was constructed are in the legend.\n\nEstimates of embryo survival from fertilisation until (A) birth or (B) four weeks after fertilisation. Numerical values derived directly from witness statements are shown as solid points. Open points have been inferred to facilitate graphical representation. Two sets of reference values have been included for comparison. The first set is derived from Table 3 of Jarvis (2016) by averaging probabilities from three independent studies. The second is from Table 4.20 of Leridon (1977). The figure clearly indicates that the extent of early embryo mortality obtained from Braude (PB/2) and emphasised by Munby J. in his judgment is substantially different from all the other witness statement estimates and those published by Jarvis and Leridon. Drife’s estimate for total pregnancy loss from fertilisation to birth is also excessive compared to the other values. The explanation for the large discrepancy in pre-implantation mortality is that Braude’s estimate is derived from in vitro laboratory and clinical IVF data, and not from natural reproduction.\n\nFour of the eight expert witnesses provided numerical estimates and/or comment on the extent of human embryo mortality: Drife, Brown, Braude and McLean. It is in these quantitative claims regarding embryo mortality that an inconsistency in the evidence and a judicial error of interpretation is apparent. I shall consider each witness statement in turn and then explain the nature and source of the scientific error in Munby J.’s judgment.\n\nProfessor Drife’s witness statement (WSJD, see Underlying data) was quoted verbatim and at length by Munby J.l, including the following section, which summarises numerically the fate of fertilised embryos:\n\n“From various strands of evidence it has been calculated that in a normally cycling woman who is sexually active and not using contraception, conception will occur in about 85% of cycles. Of those fertilised eggs, around 15% will be lost before implantation begins. Of those which begin to implant, only about half will implant successfully. Of the half which do implant successfully (as shown by detectable HCG in the woman’s urine), between one third and one half will be lost at the time of the menses. Overall, therefore, around 75% of all conceptions are followed by an apparently normal period.1”m\n\nIn the original witness statement, this passage includes a citation (Footnote 1, shown above) to a short (approx. 280 words) article7 published by Drife over 18 years previously in the British Medical Journaln. This brief article, entitled What proportion of pregnancies are spontaneously aborted? contains four citations. The first two are reviews by Short8 and Schlesselman9 that draw their quantitative conclusions ultimately from the same primary sources: namely, the unique anatomical studies of Arthur Hertig10, and French & Bierman’s observational study of 3,197 pregnancies in Kauai in the 1950s11. Henri Leridon, a renowned epidemiologist, used these same data to produce a complete life table for intra-uterine mortality in 19776. Leridon’s review has been widely cited (although not directly by Short) and his life table is reproduced as Table II in Schlesselman’s review. Strangely, despite using the same sources, Short concludes that “only about 47% of conceptions will result in a full-term live birth”8 whereas Leridon’s estimate is 37% (31/84)6. Drife’s third citation is a brief article in The Lancet by Roberts & Lowe, which concludes that 78% of all fertilised eggs perish before birth12. The fourth citation is a report by Miller et al. (1980) of a prospective study of 197 women13, estimating the loss of implanted embryos before clinical recognition of pregnancy, i.e., embryo loss between 1 and 2 weeks post-fertilisation.\n\nThese sources have been critically reviewed5. In summary: (1) Hertig’s quantitative estimates are highly imprecise; (2) French & Bierman’s study provides no relevant data regarding embryo loss during the two weeks after fertilisation; (3) Roberts & Lowe’s conclusions are derived from speculative arithmetic and have no practical quantitative value; (4) when compared to subsequent studies, Miller’s estimate of 43% loss from implantation to birth (cited by Drife) is almost certainly an overestimate.\n\nThe quantitative accounts in WSJD and the brief BMJ article partially follow Leridon (see Figure 1). The 85% fertilisation rate matches Leridon’s 84%, and derives ultimately from Hertig14. The pre-implantation loss of 15% is also similar to Leridon’s. There are however, some inconsistencies between Drife’s two accounts. For example, in the BMJ article he closely follows Leridon in stating that 15 fertilised ova fail to implant (i.e., 15/85 = 18%), but reports this as 15% (i.e., 15/100 = 15%) in WSJD. Furthermore, the conclusion in his witness statement that “75% of all conceptions are followed by an apparently normal period”o does not match the claim published in the BMJ that “the proportion of pregnancies lost after conception is 76%”p, and substantially differs from Leridon’s estimate for embryo loss before a normal period of 50%. This is principally because of the addition of an extra stage of loss between implantation and the first missed period (Figure 1). Hence, Drife overstated (perhaps inadvertently) the extent of early embryo mortality in his witness statement compared to both his own published article and the most authoritative source on which he relied.\n\nIn his 1983 BMJ article, Drife cited Miller et al. (1980) who probably exaggerate early pregnancy loss5. However, in 2001, the date of the witness statement, at least eight relevant studies on early embryo mortality had been published5, including a seminal work by Wilcox et al. (1988)15. An expert witness might have been expected to refer to some or all of these works.\n\nThis consideration of Drife’s expert testimony highlights four key points:\n\n1. Drife had written and published little on the subject of human embryo mortality.\n\n2. Published claims regarding human embryo mortality are scant, confusing and contradictory.\n\n3. Drife’s claim that 15% of embryos are lost in the first week after fertilisation before implantation is drawn from Leridon’s widely known and respected review of embryo mortality.\n\n4. Drife’s claim that 75% of all embryos are lost before an apparently normal period is an exaggeration that contradicts both Leridon’s account and his own published article!\n\nMunby J. quotes from Professor Brown’s witness statement (WSNB) only once: “It is striking that the usual fate of the fertilized egg is to die”q. Alone, this statement lacks quantitative rigour, since all fertilised eggs eventually die, the substantive issues being when and how many. However, immediately following this, in his witness statement, Professor Brown continues:\n\n“The proportion of fertilized eggs that produce a live full-term baby (in the absence of contraceptive measures) is not known precisely, but is probably only 40%1. The other 60% die, at all stages from fertilization to late pregnancy. Perhaps 20% or so do not implant in the uterus; there are no systemic signs that fertilization has occurred, and the woman is unaware. The next common stage of conceptal death is soon after implantation, when the consequence can be a heavier than usual menstrual flow, perhaps somewhat delayed, which can be noticeable.”r\n\nThis passage, from a section entitled “The Incidence of Death of Fertilised Eggs”, contains all the quantitative information on human embryo loss in WSNB. 20% loss prior to implantation is similar to the value of 15% given by Drife. Total loss of 60% from fertilisation to birth is close to Leridon’s estimate of 63%. However, the study16 providing the source for the 40% has been misinterpreted: the 60%s loss in Edmonds et al. (1982) actually indicates embryo loss from implantation and not from fertilisation as stated by Brown. Unfortunately, the data in Edmonds et al. are likely to be substantially biased owing to sup-optimal experimental design and methodology5. As noted above, more and better studies had been published by 2001 and all reported substantially lower estimates of post-implantation embryo loss.\n\nThus, in summary:\n\n1. As already noted, available scientific evidence on human embryo mortality is easily misread.\n\n2. Brown’s ball-park figure for mortality from fertilisation to birth of 60%, despite being based on a misreading of a technically biased study, is close to Leridon’s estimate of 63%. Both of these estimates are somewhat lower than Drife’s estimate of 75% loss before an apparently normal period.\n\n3. Brown’s estimate of 20% for pre-implantation loss is close to Drife’s (and Leridon’s) estimate of approximately 15%.\n\nThe interpretation of the evidence submitted by Professor Peter Braude is at the heart of the scientific misunderstanding in this case. Munby J. deliberately emphasises the extent of embryo loss as follows:\n\n“There is one other aspect of this medical evidence which perhaps requires emphasis. This is summarised by Professor Braude in the proposition that “Fertilisation does not usually result in the development of an embryo” and by Professor Brown in the statement “It is striking that the usual fate of the fertilized human egg is to die.” According to Professor Braude not much more than 25% of successfully fertilised eggs reach the blastocyst stage of development and “Even once implanted the failure rate is prodigious”, for fewer than 15% of fertilised eggs will result in a birth.”t\n\nProfessor Braude submitted to the court as evidence both a witness statement (WSPB) and a book chapter, entitled The Embryo in Contemporary Medical Science17, listed as Exhibit PB/2, jointly written and published in 1990 by him and a colleague, Professor Martin Johnsonu. The values emphasised by Munby J. in paragraph 129 of his judgment are from this book chapter. Braude does not use these values in his witness statement but merely states that “It is to be noted that of the eggs that are successfully fertilised, a large number do not eventually become implanted in the uterine wall.”v He provides no citation for this claim.\n\nUnlike Drife and Brown, Braude cites the best available study of early pregnancy loss at that time, Wilcox et al. (1988)15, stating that “nearly one quarter (22%; 43/198) of women attempting pregnancy, showed a positive hCG but did not continue to miss their menstrual period or continue with a clinical pregnancy.”w Braude also refers to another study, albeit without an explicit citation; however, it is clear from the context that the study is Ellish et al. (1996)18. He reports a post-implantation embryo loss in this study of “between 11% and 27%”x prior to the first missed period. These values are from Table VI of Ellish et al. (1996), and are consistent with the equivalent value (22%) from Wilcox et al. (1988) and many other studies5. However, it is important to note that neither Wilcox et al. (1988), Ellish et al. (1996), nor the two similar studies cited by Drife (Miller et al., 1980) or Brown (Edmonds et al., 1982) contains any data on embryo loss between fertilisation and the onset of implantation.\n\nExhibit PB/2 is an extract from a book that examines the human embryo from historical, legal and cultural perspectives. It is a scientific chapter17 and its purpose is stated clearly in the introduction: “It is our intention here to summarize as simply as possible some of our current knowledge about human early development which can serve as a basis for informed discussion.” It is a description of the formation of germ cells (sperm and ova), fertilisation, and the development and growth of the embryo up until birth. The principal focus is on the period from fertilisation up until the fetal stage, thereby reflecting the subject matter of the book as a whole. The account is very useful.\n\nThe values used by Munby J. in paragraph 129 are in one section of this chapter. Following Munby J.’s example, I shall quote the paragraph at length, underlining those phrases reproduced verbatim in his judgment:\n\n“Fertilization does not usually result in the development of an embryo. From our knowledge of human development in vitro and those limited studies of early human development in vivo, it seems that not much more than 25 per cent of successfully fertilized eggs reach the blastocyst stage of development.16 Even once implanted the failure rate is prodigious. A recent study has suggested that 22 per cent of very early pregnancies which can be detected by raised blood levels of human chorionic gonadotrophin (hCG; the hormone produced by the implanting trophectoderm) will fail.17 This group does not include those pregnancies that fail before the hCG can be produced and thus go undetected. In addition, a further 12–15 percent of clinically recognized pregnancies fail within the first 4 months of pregnancy.18 In all, fewer than 15 per cent of fertilized eggs will result in a birth.”y\n\nThis passage contains several quantitative claims. A 12–15% clinical loss is a credible estimate5. The reference to 22% is from Wilcox et al. (1988) and is followed by the important point, highlighted above, that those data do not include pregnancies that fail after fertilisation but before implantationz. In other words, that study, and by extension all studies that monitor pregnancy by detection of elevated hCG, cannot inform us about embryo mortality rates in the week after fertilisation but before implantation, that is, during the period that the MAP is typically used. The remaining two values in this passage, both reproduced in the judgment, namely, 25% survival from fertilisation to blastocyst, and fewer than 15% survival from fertilisation to birth, require further inspection.\n\nMunby J. knew that the blastocyst stage is reached prior to the commencement of implantation as indicated by his account of the physiologyaa. Table 1 in Exhibit PB/217, which Munby J. appears to have read in detail, also makes it clear that the blastocyst stage is reached at 5 days, before implantation at 7 days. Hence, it is clear that Braude & Johnson’s claim that “not much more than 25% of successfully fertilized eggs reach the blastocyst stage of development” is in stark contrast to that of Drife and Brown, that 15% or 20% of embryos are lost before implantation. According to one, only 25% survive, and according to the others, 80–85% survive to the blastocyst stage. This difference is substantial (Figure 1) and the inconsistency invites scrutiny. Did Munby J. notice this discrepancy in estimates of the same phenomenon occurring within a few paragraphs of his own judgment? Is it by chance that he chose to emphasise the one value from all the submitted evidence that maximised and exaggerated the extent of embryo mortality prior to implantation, precisely the time when the MAP is intended for use? It may be stated that if Munby J. believed that this value referred to the mortality of embryos in vivo under natural conditions, conditions under which the MAP is typically used, he was unequivocally wrong.\n\nThe basis for his error is not difficult to identify and will be clarified after considering the evidence provided by the fourth expert witness.\n\nDr McLean provided the longest written statement (WSJM1) of all the expert witnesses (Table 1). Specifically, his statement included a section entitled “Early Embryo Loss”bb comprising 740 words and 10 scientific references. (McLean provided a second witness statement (WSJM2), which addressed the issue of the dating of the commencement of pregnancy. It contains no additional information on human embryo mortality.) McLean distinguishes between different categories of study on embryo loss: those that, in principle, provide information on embryo loss before implantationcc, and those that provide information on loss only after implantation has commenceddd. In the first category, McLean discusses in some detail the unique studies of Hertig previously mentioned10. As noted with Drife and Brown, there are minor errors in McLean’s account. For example, 42 is not the maximum age of the 210 women enrolled in the study, but the maximum age of those 34 women from whom fertilised ova were recovered. More importantly, he gives three strikingly variant summary values for early embryo loss derived from Hertig: 29%, 35% and 78%.\n\nMcLean’s first value of 29% is his own calculation derived from the 10 abnormal embryos found by Hertig out of all 34 embryos recovered (10/34 = 0.29). Hertig does not use this value nor such a calculation. Making sense of Hertig’s data and calculations is not straightforward, although it is clear that only 8 of the 34 embryos recovered by Hertig were at a pre-implantation stage. An attempt to clarify his calculations has recently been published5, and argues that Hertig’s data and analytical logic indicate that 50%ee of fertilised eggs would perish up to the time of a missed menstrual period, and that 30% would perish between fertilisation and implantationff. Leridon’s equivalent values are 50% and 18%. McLean’s second value, 35%, comes from a re-interpretation of Hertig’s data by James (1970)19. However, James’ value of 35% refers to loss of all fertilised eggs before the first missed period and not just before implantation. Hence, James’ estimate is less than both Hertig’s and Leridon’s for the first two weeks after fertilisation, and he concludes that “49% of all zygotes perish naturally between fertilization and confinement”gg. McLean’s third value of 78% is from Roberts & Lowe’s Lancet article12. Roberts & Lowe cite Hertig’s work in support of their quantitative argument although it is unclear how it meaningfully informs their analysishh. Furthermore, Roberts & Lowe’s 78% estimate is not for early pregnancy loss, but for embryo loss from fertilisation to birth. Though widely cited, it is both exaggerated and unsupported by evidence.\n\nIn giving the impression that Roberts & Lowe’s estimate is derived from Hertig’s data, McLean’s account is inaccurate and unhelpful. Nevertheless, such a large numerical variance might alert an attentive reader to the difficulties associated with detecting pre-implantation human embryos, an issue McLean explicitly discussesii. In paragraphs 23–25 of WSJM1, he considers two putative biological markers, early pregnancy factor (EPF) and embryo-derived platelet activating factor (EDPAF), that had been proposed to be released within 24 hours of fertilisation. He discusses the possibility that detection of EDPAF might provide insight into the fate of embryos in the first weekjj; however, he offers no quantitative estimates from such investigations. Since 2001, little work has been published on EPF or EDPAF and any initial promise they may have had for detecting pre-implantation embryos has long since fadedkk. Munby J. was correct in stating that “The test for hCG represents the first reliable opportunity to identify the existence of an embryoll.”\n\nWSJM1 also contains results from three studies that used the detection of hCG to quantify embryo loss between implantation and a clinical diagnosis of pregnancy. The estimates were 33%13, 57%16 and 8%20 early pregnancy loss. Once again, the divergence in these values is striking and may make a thoughtful reader query their reliability. The high variance in the results from these three studies is likely to be due to limitations in experimental reagents and study design5. Given the length and detail of his statement, it is surprising that McLean does not mention Wilcox et al. (1988), the first of several studies to address these limitations. Nevertheless, McLean does make it clear that even these studies cannot, in principle, provide information on the fate of the pre-implantation embryo.\n\nMcLean also makes the important point that the extent of early embryo loss is not only a matter of biological interest, but “has acquired political significance with regard to legislation on human embryo experimentation and the use of emergency hormonal contraception”mm. Arguably, Munby J.’s emphasis on this aspect of the scientific evidence lends credence to this point. McLean continues: “It is therefore important to obtain as accurate an estimate as is possible for the occurrence of early human embryo loss.” As if to reinforce this point, he cited a study by Walker et al. (1988)21 that reported no losses of biochemical pregnanciesnn in 75 cycles and concluded that the extent of early pregnancy loss may have been “substantially overestimated.”oo This estimate of 0% loss is extreme, and a reasoned response from Wilcox argued that the results of their two studies were not necessarily inconsistent22. However, the message is clear: quantification of early embryonic loss generates confusing and highly variable results and it therefore behoves a cautious reader concerned with factual accuracy to scrutinise specific quantitative claims with care.\n\nIn emphasising that “not much more than 25% of successfully fertilised eggs reach the blastocyst stage”, a value that is quantitatively contradicted by the evidence provided by Drife, Brown and McLean (Figure 1), Munby J. reveals that he did not properly understand the significance of the expert witness evidence provided to the court on this matter. Furthermore, he cannot have examined that particular claim with due care, for if he had done, he would have discovered why the value was so low and why it was of no relevance to the case.\n\n\nMunby J.’s error\n\nIn paragraph 129 of Munby J.’s judgment there are two quantitative claims, both attributed to Professor Braude, that invite close scrutiny: (1) “not much more than 25% of successfully fertilised eggs reach the blastocyst stage of development”, and (2) “fewer than 15% of fertilised eggs will result in a birth”. Although Munby J. does not place these statements in quotation marks, they are taken directly from the book chapter submitted by Braude as evidence (Exhibit PB/2)17. In this chapter, the 25% claim is supported by a citation to a review on the early development of human embryos in vitro co-written by Dr Virginia Bolton and Professor Braude23. A “<15%” claim is found in the same review.\n\nBolton & Braude’s review of the development of the preimplantation human embryo in vitro represents IVF as “a remarkably inefficient therapeutic procedure” and explores biological and technical reasons for the “unacceptably high rate of embryonic loss” associated with IVF treatment. Within this context they describe the work of Fehilly et al. (1985)24 who…\n\n“…attempted to culture human embryos surplus to those required for replacement during therapeutic IVF cycles to the blastocyst stage for cryopreservation. Of 784 pronucleate embryos, 75% (585) were able to develop to the five- to eight-cell stage in vitro; only 34% (197) of these progressed in culture to form expanded blastocysts.”\n\n197 expanded blastocysts developing from 784 embryos is 25.1%, i.e., “not much more than 25%”.\n\nFehilly et al. (1985) conducted their study primarily to determine whether embryos frozen at the cleaving stage (day 3–4 after fertilisation) or at the blastocyst stage (day 5–6 after fertilisation) would be more effective at producing subsequent pregnancies after embryo thawing and transfer into the womb. Based on the data described above, with regard to embryo development to the blastocyst stage, they commented that “only one quarter of them expanded in vitro”. This data is the source for Munby J.’s claim that not much more than 25% of fertilised eggs reach the blastocyst stage.\n\nIn the following paragraph in their review, Bolton & Braude offer one reason for the low survival rate of the in vitro embryos: “Suboptimal culture conditions are undoubtedly responsible for a proportion of this embryonic failure”23. Elsewhere, Braude reports that “Experiments in our laboratories have suggested that the in vitro handling of oocytes can produce chromosomal aberrations at alarmingly high frequencies”25 and strikingly, that “When Bob Edwards and indeed my own group were researching these early stages, blastocyst culture was awful (about 15% of embryos made it to that stage)”26. More recently, Dr Bolton has repeated that “Embryo culture conditions in vitro are likely to be suboptimal compared with those in vivo”27. Put simply, human embryos created by fertilisation in vitro did not, and do not fare well. Hence, the use of in vitro data to define the fate of natural embryos in vivo is both biologically and quantitatively risky5.\n\nMunby J.’s second quantitative claim, that “fewer than 15% of fertilised eggs will result in a birth”, also comes from Bolton & Braude’s review via Braude & Johnson’s chapter. A further quotation from p. 93 makes the point:\n\n“In fact, IVF represents a remarkably inefficient therapeutic procedure. Although fertilization can now be achieved with consistent success in vitro, the success rate of ongoing pregnancies is much lower … If an average is taken from the longest established IVF units, it can be seen that <15% of all embryos that are replaced will result in a clinical pregnancy (Table I).”23\n\nTable I in Bolton & Braude provides a quantitative summary of the success of embryo replacement using data from seven clinical IVF units. The data are sub-divided according to whether 1, 2 or 3 embryos were replaced (i.e., transferred) into the womb of the woman undergoing treatment. The clinical pregnancy rate among the patients thus treated is reported as a percentage of embryos replaced as 12.7%, 12.1% and 9.6% respectively. These values are somewhat lower than 15% and clearly refer to clinical pregnancies. Nevertheless, this IVF data appears to be the source for the judicial claim that “fewer than 15% of fertilised eggs will result in a birth”.\n\nThere are only two other cited studies in the key paragraph of Braude & Johnson’s chapter (Exhibit PB/2)17. These are the study by Wilcox et al.15 and a paper by Professor Lesley Regan28. Wilcox et al. conclude that “The total rate of pregnancy loss after implantation, including clinically recognized spontaneous abortions, was 31 percent” and a re-analysis of that data suggests that approximately 50% of the fertilised eggs in that study may have been lost up to birth4. Regan’s paper addresses the incidence of spontaneous abortion of clinical pregnancies with a focus on recurrent abortion. It contains preliminary findings from a prospective study of pregnancy loss and, interestingly, concludes that the “overall incidence of spontaneous abortion in this prospective study is considerably lower than those reported in previous studies (10.3% overall; 5.6% for primigravidae)”28. Therefore, neither Wilcox et al. (1988) nor Regan (1987) is a credible source for Munby J.’s claim that “fewer than 15% of fertilised eggs will result in a birth”.\n\nIt is clear therefore that the two values emphasised by Munby J. in his judgment referred firstly, to embryo mortality in vitro, and secondly, to the survival of IVF embryos following their transfer into women as part of fertility treatment. It is surely reasonable to suppose that the context for the use of the MAP, subject to the judicial review, was not in vitro embryos in a laboratory or women undergoing fertility treatment, but rather naturally conceived embryos and women at risk of pregnancy. The values emphasised by Munby J. have no bearing on the case at all.\n\n\nAn understandable error?\n\nIt is charitable to assume that Munby J. did not realise that the figures he emphasised were biologically misrepresentative and therefore irrelevant to the case. Nevertheless, are there any reasons to believe that he could have spotted and avoided this error?\n\nThere are clues in Braude & Johnson’s chapter that might alert an attentive reader to the potentially misleading nature of some of its quantitative claims. In the introduction, they explicitly state that their knowledge of human development was drawn from various sources, including “studies of live preimplantation pre-embryos in vitro as part of therapeutic infertility programmes”, and in the critical paragraph from which Munby J. quotes, they preface their remarks with the phrase: “From our knowledge of human development in vitro and those limited studies of early human development in vivo”. Unfortunately, their subsequent commentary does not make it clear which of the evidence they report is from in vivo, and which from in vitro studies. Scrutiny of the references, as noted above, is revealing. Since Braude & Johnson aimed “to summarize as simply as possible some of our current knowledge about human early development”, it is perhaps understandable that they glossed over such detail. Consequently, a non-expert reader may be forgiven for concluding that their account related to normal early human development under natural conditions, particularly since no indication is given in the chapter that there are any substantive differences between in vitro and in vivo embryonic development.\n\nIt is unlikely that Braude & Johnson wrote the chapter, published in 1990, with a view to it being submitted as expert witness testimony in a judicial review in 2001, let alone that it should be directly quoted by a judge. Perhaps it would have been wise only to submit a contemporaneous witness statement addressing matters of direct relevance to the case. It would be harsh to even hint that, in submitting the chapter as expert witness evidence, Braude intended to mislead the judge on this particular matter, and it may be asking too much to expect even a learned judge to see through such a tangle of scientific evidence. Nevertheless, irrespective of whether witness, judge or both were culpable, the outcome is clear: Munby J. misjudged the extent of human embryo mortality.\n\nMunby J. had more evidence available to him than just Braude & Johnson’s chapter (Table 1). The emphasis he placed on human embryonic mortality must therefore be read in the context of that further expert witness testimony. Arguably, the contradictions between the various statements (see Figure 1) should have alerted him to these issues. Other than to acknowledge its existence, Munby J. makes no reference to McLean’s detailed and lengthy statement, prepared on behalf of the Claimant. This is regrettable, since the statement offers credible warnings about the relevance and reliability of estimates of embryo mortality in the scientific literature. The similar numerical estimates for pre-implantation loss offered independently by both Drife and Brown are in stark contrast to the one he chose to emphasise from Braude & Johnson’s chapter. Furthermore, in selectively weaving quotations from the witness testimonies, Munby J. gives the misleading impression that Braude and Brown were in agreement about the extent of early embryo mortality, despite evidence to the contrary.\n\nOne is left wondering whether Munby J. realised what he was doing.\n\n\nConclusion\n\nBraude & Johnson’s chapter was written with more than just biology in mind: the critical paragraph is in a section headed “Ethics and the biology of pre-embryos” and the conclusion touches on religious, ethical and regulatory issues. It thus appears that there was an ethical and, by extension, a legislative agenda underlying this chapter. This agenda is more explicit in a magazine article29 cited by Braude & Johnson.\n\nNatural human embryo mortality has often been linked to the ethical status of human embryos. For example, in their brief article, Roberts & Lowe state that “If Nature resorts to abortion … by discarding as many as 3 in every 4 conceptions, it will be difficult for anti-abortionists to oppose abortion on moral and ethical grounds.”12 Ronald Green, Professor Emeritus of Religion at Dartmouth College, points out, incorrectly, that “between two-thirds and three-quarters of all fertilized eggs do not go on to implant in the womb” and asks: “In view of this high rate of embryonic loss, do we truly want to bestow much moral significance on an entity with which nature is so wasteful?”30 A report of the Ethics Committee of the Royal College of Obstetricians and Gynaecologists in 1983 states: “Knowing as we do that in the natural process large numbers of fertilised ova are lost before implantation, it is morally unconvincing to claim absolute inviolability for an organism with which nature itself is so prodigal”31. This link has been considered by many others32–34. Thus, McLean’s assertion, in evidence, that early embryo loss is not only of biological interest but also of political and legislative significancepp, was clearly correct. How specific estimates of embryo mortality inform an ethical calculus is, perhaps, not so clear. Nevertheless, for those who consider it germane, McLean’s exhortation that “It is therefore important to obtain as accurate an estimate as is possible for the occurrence of early human embryo loss”, must surely be correct too.\n\nDid the quantitative bias in Munby J.’s description of embryo mortality have a significant influence on his legal judgment? If not, why then, one may ask, in such a “very long” judgment, would he deliberately choose to emphasise this particular point? What purpose might such an observation serve? Perhaps these are questions for other, legal minds. However, the biology is a different matter. Judgments carry weight and can influence opinion. Even on biological matters, legal scholars may rely upon the sayings of learned judges rather than scientific evidence.\n\nIn the first four editions of her popular undergraduate textbook35–38, Emily Jackson discusses Munby J.’s judgment and comments on embryo loss: “Approximately 75 per cent of all naturally fertilized eggs will be lost before the woman’s next period, and it would be counterintuitive to describe these losses as miscarriages”qq. Although she does not offer the judgment as her source, her words closely reflect Drife’s evidence, repeated verbatim in the judgmentrr. Her addition of the word “naturally” makes explicit the implied, and incorrect, sense in the judgment. It is notable that in the most recent (2019) edition of her book39 the explicit 75% claim is omitted: “The majority of naturally fertilized eggs will be lost before the woman’s next period, and it would be counterintuitive to describe these losses as miscarriages.”ss\n\nAnother legal scholar, Jonathan Herring, in all eight editions of his textbook40–47, quotes directly from paragraph 129 of Munby J.’s judgment in order to outline an ethical argument:\n\n“Those who disagree with the argument that personhood begins at conception … could also make the following argument: ‘[It] is striking that the usual fate of the fertilized human egg is to die.’† It has been estimated that fewer than 15 per cent of fertilized eggs will result in a birth.§”tt\n\nThe first reference (†) correctly attributes these words to Professor Brown, as found in the judgment. Strangely, the second reference (§) is to an article by Professor John Harris on assisted reproductive technological blunders48 that has no bearing on the issue. Clearly, it is a direct quotation from Braude & Johnson (1990)17 and paragraph 129 of Munby J.’s judgment.\n\nIt is unfortunate that these biological errors have found their way into standard legal textbooks. Irrespective of the philosophical merits of arguments such as those outlined above, most would agree that in order to arrive at defensible ethical and legal conclusions it is necessary to begin with reliable and relevant evidence. It would be helpful if Munby J. were to clarify whether he realised that the numerical estimates he emphasised in paragraph 129 of his judgment were derived from in vitro circumstances, and were therefore not representative of natural in vivo situations. It would also be instructive to know whether Munby J. still believes that “not much more than 25% of successfully fertilised eggs reach the blastocyst stage of development”. If the answers to these queries are negative, a clarification from him regarding the biology would be welcome, in the hope that the widely held, unsubstantiated and excessively pessimistic view of natural human embryo survival may, little by little, be replaced with one that is more closely based on available, relevant, scientific evidence5.\n\n\nData availability\n\nApollo - University of Cambridge Repository: UnderlyingData_MisjudgingEarlyEmbryoMortality_Redacted, https://doi.org/10.17863/CAM.536963\n\nThis dataset consists of transcripts of scientific witness statements submitted as evidence in R (on the application of Smeaton) v Secretary of State for Health [2002] EWHC 610 (admin) (18 April 2002) (Case No: CO/928/01). Some content has been redacted, including personal addresses.\n\nCopyright (“all rights reserved”) for the content of the witness statements remains with the authors of the statements.\n\nPhotocopies of original expert witness statements were obtained from the archives of the Claimant. A full unredacted (except for personal addresses) transcript and copies of the statements are available from the author on request.\n\n\nNotes\n\na Paragraphs from the judgment (and witness statements) are referenced in footnotes as follows: Munby [1]. The full judgment is available here: http://www.bailii.org/ew/cases/EWHC/Admin/2002/610.html\n\nb Munby [346].\n\nc Munby [2].\n\nd The Civil Procedure Rules, r.32.12(2)(c)\n\ne The Civil Procedure Rules, r.31.22(1)(a)\n\nf Munby [32].\n\ng Munby [125] & [191].\n\nh Munby [32].\n\ni Munby [126].\n\nj Munby [126(iii)].\n\nk Munby [126(ix)].\n\nl Munby [131-5] & [137]; WSJD [3-4], [8], [11-12] & [16].\n\nm Munby [134]; WSJD [8]. Footnote 1 (shown at the end of the quotation) is found in WSJD but omitted from the judgment, and reads as follows: “1 Drife, JO. British Medical Journal 1983; 286:294.”\n\nn The BMJ has no record of whether this response to a reader question (or similar short items) would have been peer-reviewed in 1983 (personal email correspondence from BMJ editorial office, 23rd June 2020).\n\no WSJD [8]. This claim only makes sense if his estimate of post-implantation loss of ‘between one third and one half’ is interpreted as 42% (average of 33% and 50%). However, in his BMJ piece, Drife states that out of 36 women with detectable hCG, only 24 will miss a period, indicating a loss at this stage of 12/36 = 33%. Thus, his estimate of post-implantation loss is inflated in WSJD compared to the BMJ.\n\np The BMJ figure incorporates an estimate (‘between 10% and 30%’) for the rate of pregnancy loss after a woman knows she is pregnant. To be internally consistent in the BMJ piece, a clinical pregnancy failure rate of 15% must be used. Applying this estimate to WSJD results in a total loss between fertilisation and birth of 79%.\n\nq Munby [129]; WSNB [22].\n\nr WSNB [22]. Footnote 1 in this passage reads as follows: “Edmonds D.K., Lindsay K.S., Miller J.F., Williamson E., Wood P.J. Early Embryonic Mortality in Women, Fertility and Sterility 1982 Vol 38 447–453”.\n\ns The precise value reported by Edmonds et al. (1982) was 61.9%.\n\nt Munby [129].\n\nu It is both likely and appropriate that Munby J. would have regarded Professor Johnson also as a “very eminent medical expert”.\n\nv WSPB [8].\n\nw WSPB [15]. Braude’s description of this data is slightly inaccurate. The study followed 221 women who were attempting to get pregnant, and the value of 22% (43/198) actually refers to the number of hCG positive menstrual cycles (198 out of 707 monitored cycles) that did not manifest as a clinical pregnancy, i.e., 43. 22% therefore refers to embryo loss from the onset of implantation (as indicated by the positive hCG test) up to, but not including, clinical diagnosis (as indicated by an absent menstrual period or subsequent positive pregnancy test).\n\nx WSPB [15].\n\ny Braude & Johnson (1990), p. 218. The passage contains three endnotes as follows:\n\nEndnote 16 reads: “V. N. Bolton, and P. R. Braude, in A. McLaren and G. Siracusa, eds, Current Topics in Developmental Biology, Vol 23, Recent advances in mammalian development, Academic Press, 1987, pp. 93-114.”\n\nEndnote 17 reads: “A. J. Wilcox, C. R. Weinberg, J. F. O’Connor, D. D. Baird, J. P. Schlatterer, R. E. Canfield, E. G. Armstrong and B. C. Nisula, Incidence of early loss of pregnancy’, New Eng. J. Med., 319, 1988, 189-94.”\n\nEndnote 18 reads: “L. Regan ‘A prospective study of spontaneous abortion’, in: R. W. Beard and F. Sharp, eds, Early Pregnancy Loss, 18th Study group of the Royal College of Obstetricians and Gynaecologists, Springer-Verlag, 1987, pp. 23-37.”\n\nz Given the matter under consideration in the judicial review, it is notable that, in this passage, Braude & Johnson clearly use the word ‘pregnancies’ to refer to women carrying a fertilised egg before implantation.\n\naa Munby [126(vii)] & [126(viii)].\n\nbb WSJM1 [30–34].\n\ncc WSJM1 [31] & [33].\n\ndd WSJM1 [32].\n\nee This value of 50% ultimately derives from the 8 pre-implantation embryos, of which 4 were abnormal and therefore assumed to be destined to perish before the end of the first 2 weeks after fertilisation. It is therefore based on a very small sample size.\n\nff It is a numerical coincidence that McLean’s value of 29% (10/34) is so close to this value of 30%.\n\ngg A zygote is the newly fertilised ovum: a one-cell embryo. Confinement is the time of childbirth.\n\nhh For example, Roberts & Lowe use Hertig’s work as a source for their claim that coitus at the time of ovulation results in a fertilisation rate of 50%. This contrasts with Hertig’s own conclusion, followed by Leridon, that ‘when conditions are optimal about 15 per cent of oocytes fail to become fertilized’ (Hertig, 1967).\n\nii WSJM1 [33].\n\njj WSJM1 [33].\n\nkk A PubMed (https://www.ncbi.nlm.nih.gov/pubmed) search on <\"early pregnancy factor\"[All Fields] NOT Review[ptyp]> performed on 3rd June 2020 identified 142 articles published between 1977 and 2000, and 35 from 2001 to the present day. A search on <“embryo derived platelet activating factor\"[All Fields]> identified only 26 articles, published between 1985 and 2004. Only two of these were published after 1992.\n\nll Munby [126(ix)].\n\nmm WSJM1 [34].\n\nnn “Biochemical pregnancy”: this term is sometimes used to refer to pregnancies that are detected solely by elevated hCG rather than by clinical observations such as a missed menstrual period.\n\noo This phrase is found both in WSJM1 [34] and the abstract of Walker et al. (1988).\n\npp WSJM1 [34].\n\nqq Jackson, E., Medical Law: Text, Cases, and Materials. 1st ed. (2006), p. 624; 2nd ed. (2010), p. 693; 3rd ed. (2013), p. 703; 4th ed. (2016), p. 737.\n\nrr “…around 75% of all conceptions are followed by an apparently normal period. These losses … are not covered by the term ‘miscarriage’.” from WSJD [8] and Munby [134].\n\nss Jackson, E., Medical Law: Text, Cases, and Materials. 5th ed. (2019), p. 770.\n\ntt Herring, J., Medical Law and Ethics. 1st ed. (2006), p. 250; 2nd ed. (2008), p. 285; 3rd ed. (2010), p. 310; 4th ed. (2012), p. 318; 5th ed. (2014), p. 320; 6th ed. (2016), p. 332; 7th ed. (2018), p. 331; 8th ed. (2020), p. 374.", "appendix": "References\n\nR (on the application of Smeaton) v Secretary of State for Health, [2002] EWHC 610 (Admin), [2002] All ER (D) 115 (Apr) (2002). Reference Source\n\nNAB v Serco Ltd & Anor, [2014] EWHC 1225 (QB) (2014). Reference Source\n\nJarvis G: UnderlyingData_MisjudgingEarlyEmbryoMortality- Redacted. Dataset. Apollo - University of Cambridge Repository: http://www.doi.org/10.17863/CAM.53696\n\nJarvis GE: Estimating limits for natural human embryo mortality [version 2; referees: 2 approved]. F1000Res. 2016; 5: 2083. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJarvis GE: Early embryo mortality in natural human reproduction: What the data say [version 2; peer review: 2 approved, 1 approved with reservations]. F1000Res. 2017; 5: 2765. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeridon H: Intrauterine Mortality. Human Fertility: The Basic Components. Chicago: The University of Chicago Press; 1977; 48–81. Reference Source\n\nDrife JO: What proportion of pregnancies are spontaneously aborted? Brit Med J. 1983; 286(6361): 294. Reference Source\n\nShort RV: When a conception fails to become a pregnancy. Ciba Found Symp. 1978; (64): 377–94. PubMed Abstract \| Publisher Full Text\n\nSchlesselman JJ: How does one assess the risk of abnormalities from human in vitro fertilization? Am J Obstet Gynecol. 1979; 135(1): 135–48. PubMed Abstract\n\nHertig AT, Rock J, Adams EC, et al.: Thirty-four fertilized human ova, good, bad and indifferent, recovered from 210 women of known fertility; a study of biologic wastage in early human pregnancy. Pediatr. 1959; 23(1 Part 2): 202–11. PubMed Abstract\n\nFrench FE, Bierman JM: Probabilities of fetal mortality. Public Health Rep. 1962; 77: 835–47. PubMed Abstract \| Free Full Text\n\nRoberts CJ, Lowe CR: Where have all the conceptions gone? Lancet. 1975; 305(7905): 498–9. Publisher Full Text\n\nMiller JF, Williamson E, Glue J, et al.: Fetal loss after implantation. A prospective study. Lancet. 1980; 316(8194): 554–6. PubMed Abstract \| Publisher Full Text\n\nHertig AT: The Overall Problem in Man. In: Benirschke K.,editor. Comparative Aspects of Reproductive Failure: An International Conference at Dartmouth Medical School. Berlin: Springer Verlag; 1967; 11–41. Publisher Full Text\n\nWilcox AJ, Weinberg CR, O'Connor JF, et al.: Incidence of early loss of pregnancy. N Engl J Med. 1988; 319(4): 189–94. PubMed Abstract \| Publisher Full Text\n\nEdmonds DK, Lindsay KS, Miller JF, et al.: Early embryonic mortality in women. Fertil Steril. 1982; 38(4): 447–53. PubMed Abstract\n\nBraude PR, Johnson MH: The Embryo in Contemporary Medical Science. In :Dunstan GR., editor. The Human Embryo: Aristotle and the Arabic and European Traditions. Exeter: University of Exeter Press; 1990; 208–21. Reference Source\n\nEllish NJ, Saboda K, O'Connor J, et al.: A prospective study of early pregnancy loss. Hum Reprod. 1996; 11(2): 406–12. PubMed Abstract \| Publisher Full Text\n\nJames WH: The incidence of spontaneous abortion. Popul Stud (Camb). 1970; 24(2): 241–5. PubMed Abstract \| Publisher Full Text\n\nWhittaker PG, Taylor A, Lind T: Unsuspected pregnancy loss in healthy women. Lancet. 1983; 1(8334): 1126–7. PubMed Abstract \| Publisher Full Text\n\nWalker EM, Lewis M, Cooper W, et al.: Occult biochemical pregnancy: fact or fiction? Br J Obstet Gynaecol. 1988; 95(7): 659–63. PubMed Abstract \| Publisher Full Text\n\nWilcox AJ, Weinberg CR, Baird DD: Subclinical embryonic loss. Fertil Steril. 1989; 51(5): 907–8. PubMed Abstract \| Publisher Full Text\n\nBolton VN, Braude PR: Development of the human preimplantation embryo in vitro. Curr Top Dev Biol. 1987; 23: 93–114. PubMed Abstract \| Publisher Full Text\n\nFehilly CB, Cohen J, Simons RF, et al.: Cryopreservation of cleaving embryos and expanded blastocysts in the human: a comparative study. Fertil Steril. 1985; 44(5): 638–44. PubMed Abstract \| Publisher Full Text\n\nBraude PR, Johnson MH, Pickering SJ, et al.: Mechanisms of Early Embryonic Loss In Vivo and In Vitro. In: Chapman. M, Grudzinskas G, Chard T, editors. The Embryo: Normal and Abnormal Development and Growth. London: Springer-Verlag; 1991; 1–10. Publisher Full Text\n\nBraude P: Selecting the 'best' embryos: prospects for improvement. Reprod Biomed Online. 2013; 27(6): 644–53. PubMed Abstract \| Publisher Full Text\n\nBolton VN, Leary C, Harbottle S, et al.: How should we choose the 'best' embryo? A commentary on behalf of the British Fertility Society and the Association of Clinical Embryologists. Hum Fertil (Camb). 2015; 18(3): 156–64. PubMed Abstract \| Publisher Full Text\n\nRegan L: A prospective study of spontaneous abortion. In: Beard RW, Sharp F, editors. Early Pregnancy Loss: Mechanisms and Treatment: Springer-Verlag; 1987; 23–37. Publisher Full Text\n\nJohnson M: Did I begin? New Sci. 1989; 124(1694): 39–42. PubMed Abstract\n\nGreen RM: The Human Embryo Research Debates: Bioethics in the Vortex of Controversy. Oxford: Oxford University Press; 2001. Reference Source\n\nLoudon JDO, Butcher CHH, Dunstan GR, et al.: Report of the RCOG Ethics Committee on In Vitro Fertilisation and Embryo Replacement or Transfer. London: 1983. Reference Source\n\nOrd T: The scourge: Moral implications of natural embryo loss. Am J Bioethics. 2008; 8(7): 12–9. PubMed Abstract \| Publisher Full Text\n\nHarris J: Stem cells, sex, and procreation. Camb Q Healthc Ethics. 2003; 12(4): 353–71. PubMed Abstract \| Publisher Full Text\n\nGreasley K: In Defense of Abortion Rights. In: Greasley K, Kaczor C, editors. Abortion Rights: For and Against. Cambridge: Cambridge University Press; 2018; 1–85. Publisher Full Text\n\nJackson E: Medical Law: Text, Cases, and Materials. 1st ed: Oxford University Press; 2006. Reference Source\n\nJackson E: Medical Law: Text, Cases, and Materials. 2nd ed: Oxford University Press; 2010. Reference Source\n\nJackson E: Medical Law: Text, Cases, and Materials. 3rd ed: Oxford University Press; 2013. Reference Source\n\nJackson E: Medical Law: Text, Cases, and Materials. 4th ed: Oxford University Press; 2016. Reference Source\n\nJackson E: Medical Law: Text, Cases, and Materials. 5th ed: Oxford University Press; 2019. Reference Source\n\nHerring J: Medical Law and Ethics. 1st ed. Oxford: Oxford University Press; 2006.\n\nHerring J: Medical Law and Ethics. 2nd ed. Oxford: Oxford University Press; 2008. Reference Source\n\nHerring J: Medical Law and Ethics. 3rd ed. Oxford: Oxford University Press; 2010.\n\nHerring J: Medical Law and Ethics. 4th ed. Oxford: Oxford University Press; 2012. Reference Source\n\nHerring J: Medical Law and Ethics. 5th ed. Oxford: Oxford University Press; 2014. Reference Source\n\nHerring J: Medical Law and Ethics. 6th ed. Oxford: Oxford University Press; 2016. Reference Source\n\nHerring J: Medical Law and Ethics. 7th ed. Oxford: Oxford University Press; 2018. Reference Source\n\nHerring J: Medical Law and Ethics. 8th ed. Oxford: Oxford University Press; 2020. Reference Source\n\nHarris J: Assisted reproductive technological blunders (ARTBs). J Med Ethics. 2003; 29(4): 205–6. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "67635", "date": "11 Aug 2020", "name": "Maureen L. Condic", "expertise": [ "Reviewer Expertise Human embryology", "Developmental neuroscience", "Human stem cell biology", "Bioethics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 “Misjudging early embryo mortality in natural human reproduction” considers the evidence presented in a 2002 court case [R (on the application of Smeaton) v Secretary of State for Health], involving the use of Levonelle (the ‘morning after pill’; MAP) as a form of birth control. In this case, it was ruled that MAP does not act to terminate a pregnancy, a conclusion that relied heavily on the assertion that only 25% of successfully fertilized eggs survive to implantation. Here, the scientific and medical evidence in support of this assertion is examined, based on the published literature and the testimony of the four expert witnesses who provided numerical estimates of embryo mortality. In particular, the figures relied on in the Judicial opinion are derived from studies of embryo survival during IVF, and are therefore irrelevant to the use of MAP as birth control.\n\nThe argument is clearly written and provides ample evidence in support of the author’s conclusion that the case was decided, based in part, on a misinterpretation of the scientific evidence. The author points out the need for a legal analysis to clarify the impact of this error, yet (appropriately) does not offer an opinion on this matter.\n\nOver all, this is a helpful and clear critique of a seminal case involving human embryo mortality.\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": "74206", "date": "16 Nov 2020", "name": "Nicanor Austriaco, OP", "expertise": [ "Reviewer Expertise Molecular and cellular biology. Bioethics." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, Gavin Jarvis examines the scientific and medical evidence that was presented by expert witness testimony during a judicial review conducted by the Honorable Mr. Justice Munby of the administrative court in 2002 to determine whether supplying the morning after pill (MAP) is comparable to the procuring of a poison for the purpose of an abortion. If so, the claimant argued that the MAP should be regulated in the same way as any other abortion, as required by the Abortion Act 1967. Justice Munby ruled against the claimant, relying heavily on expert witness testimony that claimed that 75% of human embryos that are conceived are lost during pregnancy. Jarvis interrogates each of the witness testimonies in great detail and provides a genealogy of each of the scientific and medical claims. He successfully shows that the available data do not support the conclusion that human embryo mortality is high at 75%.\nThe claims and conclusions made by the author are validated by sound reasoning. He provides frequent citations to the primary literature to support his argument that the expert witness testimonies were often inaccurate or inconsistent. As Jarvis himself acknowledges, it would be helpful for someone to determine the impact of his analysis on the law.\nIn sum, this essay is a robust and helpful critique of a legal precedent that relied heavily on weak scientific and medical data to claim that human embryo mortality is astronomically high.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-702
https://f1000research.com/articles/9-700/v1	13 Jul 20	{ "type": "Research Article", "title": "Relationship between neighbourhood cohesion and disability: findings from SWADES population-based survey, Kerala, India", "authors": [ "M.D. Saju", "Anuja Maria Benny", "Komal Preet Allagh", "Binoy Joseph", "Jotheeswaran Amuthavalli Thiyagarajan", "Anuja Maria Benny", "Komal Preet Allagh", "Binoy Joseph", "Jotheeswaran Amuthavalli Thiyagarajan" ], "abstract": "Background: The burden of disability on individuals and society is enormous in India, and informal care systems try to reduce this burden. This study investigated the association between neighbourhood cohesion and disability in a community-based population in Kerala, India. To the best of our knowledge, no previous studies have examined this association in India.\n\nMethods: A cross-sectional household survey was conducted with 997 participants aged 30 years and above, in Kerala. Neighbourhood cohesion was assessed by three scales: trust, community participation, and perceived safety. Functional ability was measured by WHODAS 2.0. Explanatory covariates included chronic disease conditions, age, gender, education, income, and mental health conditions. Results: Of 997 participants (37% male; mean age, 53.9 [range, 30–90] years), the majority were married or cohabiting. Univariate analysis showed functional ability to be positively associated with most demographic and health characteristics. However, after adjustment, only social cohesion, age, income, education, chronic diseases and mental health conditions remained significant. Mediation analysis showed the effect of personal and health characteristics on functional ability as mediated by social cohesion. Conclusion: Social cohesion is an important moderator of functional ability. Interventions targeting the creation of stronger ties among neighbours and a sense of belonging should be scaled-up and evaluated in future research.", "keywords": [ "Neighbourhood", "Social cohesion", "Disability", "Population study", "India" ], "content": "Introduction\n\nOf the one billion people living with some form of disability in the world, nearly 200 million experience considerable difficulties in physical functioning (WHO, 2011). The International Classification of Functioning, Disability and Health defines disability as an umbrella term for impairments, activity limitations and participation restrictions (WHO, 2002). Disability appears to be a biological and social phenomenon, but the person with impairment faces multifaceted issues in various domains of life, that prevent them from full participation in society (WHO, 2011). Social exclusion prevents people with disability accessing various formal and informal services, such as health care, education, employment, social services, housing and transport (WHO, 2015). Disability is a development issue because persons with disabilities experience increased poverty and deprivation due to the above mentioned barriers compared with persons without disabilities (WHO, 2011). Poverty and deprivation is a predictor of increased impairments through malnutrition, poor access to health, and deprived living, working and travelling conditions. Disability creates a vicious cycle of poverty through lack of access to education and employment, and through increased out of pocket expenditure (OOP). Even a small OOP expenditure on health can put them at risk of drifting to below poverty line, especially for households that are just above the poverty line (van Doorslaer et al., 2006). Though the life expectancy of the general population has increased due to education, per capita income, living conditions and medical practices (Wang et al., 2017), life expectancy has not increased in the same extent for people with disabilities (Murray et al., 2015). Therefore, reducing morbidity is paramount for improving wellbeing of the population, as well as attaining the United Nations Sustainable Development Goals.\n\nParallel to epidemiological transitions, India, particularly the state of Kerala, is experiencing social, cultural and economic transitions. The key factors driving these include changes in family structures, urbanization and industrialization. These unprecedented changes influence neighbourhood cohesion, social interactions and support systems of the entire community. Several earlier studies have found a positive impact of neighbourhood social cohesion on disability (Avlund et al., 2004; Fiddian-Qasmiyeh et al., 2014). Social cohesion is defined as a cohesive society that works towards the well-being of all its members, fights exclusion and marginalization, creates a sense of belonging, promotes trust, and offers its members opportunity of upward mobility (Laiglesia, 2012).\n\nA putative explanation for neighbourhood is often referred to as a person in a situation that includes availability of all to facilities and provisions including health care, formal and informal services, and safe, secure and supportive social engagement opportunities. There are a number of studies that have investigated the effect of neighbourhood social interaction on disability and found that neighbourhood social cohesion, the perceived degree of connectedness among neighbours and their willingness to intervene for the common good, is an important aspect (Avlund et al., 2004; Fiddian-Qasmiyeh et al., 2014). Beyond that, the impact of neighbourhood cohesion and social interaction on disability and common mental issues like depression, anxiety and stress is less understood. In this study, we aim to study the association of a person’s disability with their social cohesion, and explore the channels through which disability affects social cohesion. We conducted a search on electronic databases such as, Medline, psychINFO and PubMed, using MeSH terms (social cohesion social support, disability, India), which yielded no results, concluding that no such studies have been done previously in India, to the best of our knowledge.\n\nIn the last three decades, India has experienced a major demographic, health, social and economic transitions and its impact in social cohesion has not been systematically looked at. In the context of paucity of evidence for social cohesion, this study investigates the relationship between social cohesion and functional ability after accounting for all known confounding factor,; and to what extent the effect of personal and health characteristics on functional ability is mediated by social cohesion.\n\n\nMethods\n\nThe present study is based on data collected during the ‘Social well-being and determinants of health’ SWADES study Families of the SWADES cohort were invited to be part of the baseline questionnaire between April and May 2018 (Saju et al., 2020a). The study catchment area was located in semi-rural region of Keezhmadu panchayat in Ernakulam, Kerala, India. The residents had mixed culture and socioeconomic background. Catchment area boundaries were precisely defined. Mapping was carried out to identify and locate all households. All the family members residing in that geographically located area aged 30 years and above were considered for the study. The objectives of the SWADES study are as follows: a) to monitor changes over time in physical, behavioural and social risk factors associated with chronic diseases and mental health comorbid conditions; b) to develop chronic disease risk prediction models and estimate the probability of having or developing a particular chronic disease within a specified period; and c) to scale up population and family health interventions and evaluate the impact. SWADES collected data pertaining to sociodemographic, physical, mental, functional, social cohesion, social support networks, behavioural risk factors, health services utilisation, cognitive function and risk of fall using respective scales or tools. A detailed description of the SWADES study and the recruitment of the sample population is presented in the SWADES protocol paper (Saju et al., 2020b).\n\nParticipants provided written informed consent and the study was approved by the Institutional Review Board of Rajagiri Hospital (Study Reference Number: RAJH 18003).\n\nThe SWADES baseline data was used for the present study, and this study covered only two aspects of the SWADES full survey – Functional Ability and Social Cohesion.\n\nWe used a core minimum data set with cross-culturally validated assessments (mental conditions, physical health, demographics, extensive non-communicable disease risk factor questionnaires, disability/functioning and health service utilization). All study instruments were translated, back-translated, and assessed for acceptability and conceptual equivalence. Translations were done locally, by investigators fluent in English (the language of the instruments) and in the local language (Malayalam) to be used in the study. The local version was reviewed by experts in Kerala.\n\nSociodemographic and health conditions. Education level was ascertained, and coded as: no education, did not complete primary, completed primary, completed secondary and completed tertiary education. Type of family was described as: living alone, nuclear family, extended family and mixed family. Income was a continuous variable and was coded as quartile. We also recorded age, sex, marital status (single, married/cohabitating or divorced/widowed/separated), current occupational status, and family income.\n\nSelf-reported health conditions, such as stroke, diabetes, hypertension, heart disease, were reported by participants.\n\nMeasures of functional ability. The functional ability of each participant was measured by the World Health Organization Disability Assessment Schedule 2.0 (WHODAS 2.0), measuring health and disability (Marti & Choi, 2020). The instrument covers six domains: 1) understanding and communicating with the world; 2) moving and getting around; 3) self-care; 4) getting along with people; 5) life activities; and 6) participation in society. Scores for each question range from 0 (no difficulty) to 4 (extreme difficulty/cannot do). The standardized global score ranges from 0 (non-disabled) to 100 (maximum disability) and this score was divided into quintiles. This measurement has been extensively validated in India and other low and middle-income countries (Thomas et al., 2016).\n\nMeasures of social cohesion. Social cohesion was measured from the inner to the outer social circle of the participants through three items, including trust in neighbours and co-workers, community participation, perceived safety in the participant’s residential neighbourhood and general social trust. These three items were measured using a scale (see below) that has been extensively used in research conducted in low and middle income countries (Fernández-Niño et al., 2019; Kulkarni & Shinde, 2015; Ramlagan et al., 2013).\n\nTrust in neighbours and co-workers: The scale was standardized for convenience of interpretation. The subject responded to three questions about trust according to the response options ‘to a very great extent’, ‘to a great extent’, ‘neither great nor small extent’, ‘to a small extent’ and ‘to a very small extent’:\n\nTrust in people in the participants’ neighbourhood;\n\nTrust in people with whom the participants work; and\n\nTrust in strangers.\n\nCommunity participation: This item was measured with a standardized scale used in the Social Cohesion section of the SAGE questionnaire (Kulkarni & Shinde, 2015), which indicates the frequency of involvement in community activities in the last 12 months. The subject responded to nine activities with the response options ‘never’, ‘once or twice per year’, ‘once or twice a month’, ‘once or twice a week’ or ‘daily’:\n\nAttending public meetings in which there was discussion of local or school affairs;\n\nMeeting personally with a community leader;\n\nAttending any group, club, society, union, or organization meeting;\n\nWorking with people in the neighbourhood to fix or improve something;\n\nHaving friends over to one’s home;\n\nBeing in the home of someone who lives in a different neighbourhood;\n\nSocializing with co-workers outside of work;\n\nAttending religious services (excluding weddings and funerals); and\n\nGetting out to attend social meetings, activities, programs, or events or to visit relatives or friends.\n\nPerceived safety in the participant’s residential neighbourhood: This was assessed based on two questions. The respondent answered to each according to the response options ‘completely safe’, ‘very safe’, ‘moderately safe’, ‘slightly safe’ or ‘not safe at all’:\n\nHow safe from crime and violence the participant feels when he or she is alone at home; and\n\nHow safe the respondent feels when walking down his/her street alone after dark.\n\nAll three variables were computed separately and standardized versions of three variables, trust, safety and participation were created using ‘egen’ command in STATA. Value range was then defined from 0 to 30. Based on this score, social cohesion was divided into five quintiles with lowest value in Quintile 1 (Q1) and highest value in Quintile 5 (Q5). Respondents in Q1 has lowest Social Cohesion and respondents in Q5 has highest Social Cohesion.\n\nAll statistical analysis was performed using STATA 14 and R version 3.6.3 We first compared the demographic characteristics of study participants using chi square tests, t-tests or Wilcoxon rank-sum tests, as appropriate to evaluate statistical significance. To evaluate the variables associated with the outcome of interest, we performed linear regression and calculated 95% confidence interval, and p-values to evaluate the statistical significance. Using R software, we plotted a jitter plot to study the association of disability with social cohesion. Finally, we performed path analysis to develop a hypothesis on probable causal relationship and recommendations for intervention development.\n\n\nResults\n\nWe interviewed 997 participants, aged 30 years and above and residing in 573 households of the study area. The mean age of the population was 53.9 ± 14.2 years. The majority were married or cohabiting (82.7%). More than half (54%) of the population had completed primary education. Almost all (89%) the participants were born in a village. More than half of the women were housewives (50.5%) compared to 5% of men who were house husbands. Men who were employed with paid work were triple the number of women (61.4% vs. 18.5%), highlighting a patriarchal societal set-up. Two-fifths (41.8%) of the population had very low income. More than half (60.9%) of the population resided in a nuclear family (nuclear family consists of a couple and their dependent children; higher percentage of nuclear family imply lesser number of family carers for the care of people with disability, especially in the Indian context). The percentage distribution of the sample by selected socio-demographic variables and gender is presented in Table 1.\n\nAmong the study population, the prevalence of disability based on the WHO DAS score was 19.2%. Univariate analysis was employed to identify the crude association between disability and other exploratory variables (Table 2). The prevalence of disability is twice the amount in women as compared to men (23.9% versus 11.0%). Disability prevalence increased with increase in age, lack of formal education, and presence of one or more physical or mental health condition. Participants who were widowed, divorced or separated had a higher prevalence of disability compared to married or unmarried participants. The disability scores were higher for the participants with lower economic status.\n\nThere was significant negative association between social cohesion and disability (-5.8 (-7.7 to -4), p=0.000). It was also observed that there was an independent association between people’s trust in neighbourhood (-0.57 (-0.83 to -0.30), p<0.001) and community participation (-0.38 (-0.47 to -0.29), p<0.001) with the disability.\n\nThe mean social cohesion score was 17.04 (± 6.70). Men had a higher chance of social cohesion compared to women (1.13 (0.80 to 1.47); p<0.001). Those above the age of 70 were found to have lower social cohesion compared to the other age groups (-1.04 (-1.61 to -0.47); p<0.001). While looking into the marital status of the respondents, married respondents had a socially active life compared to unmarried, widowed or divorced (1.37 (0.14 to 2.59); p=0.029). Compared to those who had no formal education, individuals with formal education took part in more social activities (1.27 (0.44 to 2.10), p= 0.003).\n\nFigure 1 shows the association of disability with social cohesion. It shows that individuals with higher disability score reside in neighbourhoods with low social cohesion.\n\nMultivariate analysis was performed to understand the association between social cohesion and disability after controlling for the confounding variables age, gender, education, marital status, occupation, income, physical condition and mental health condition (Table 2). With the influence of the confounding variables, the coefficient value was reduced, but the results remained statistically significant. There was still a strong negative association between social cohesion and disability. This indicates that individuals with lower participation in the community, trust and safety exhibited higher levels of disability.\n\nStructural model: First, we tested the direct effect of social cohesion (predictor variable) on disability (dependent variable) without mediators. The directly standardized path coefficient was not significant. Subsequently, the mediation model was tested, which included three mediators of mental health (depression, anxiety and stress) and a direct path from social cohesion to disability. The results showed that the model was a very good fit to the data. After adding the demographic variables, such as age, gender, income, education, as shown in Figure 2, the mediation model was tested again. The final meditation model showed a very good fit to the data.\n\nTaken together, these results show the important role of social cohesion in the relationship between mental health (depression, anxiety and stress) and disability. The effect of social cohesion on disability through mental health is very high. The model revealed that demographic variables, such as, age, income, education and mental health, had a direct effect on disability. In addition, social cohesion had an indirect effect on disability through mental health.\n\n\nDiscussion\n\nTo the best of our knowledge, this is the first study to examine the association between neighbourhood cohesion and disability among a community-based population in Kerala, India. In the overall sample, we found a significant association between social cohesion and disability, which suggested an increase in neighbourhood social cohesion would decrease the chance of disability. This association was pertinent even after controlling for a number of confounding variables. This study suggests that neighbourhood social cohesion may exert an effect on Indian population’s disability status.\n\nThe findings of our study are consistent with previous studies and add to the evidence. Existing literature confirms that low social cohesion increases poor self-rated health and long-term disability (Avlund et al., 2004; Hyyppä & Mäki, 2001; Laiglesia, 2012; Pampalon et al., 2007). Previous studies found women to be more responsive to neighbourhood environmental influences because of lower levels of labour market participation and higher levels of family care and time spent at home, as opposed to men who may be spending more time away from the neighbourhood (Patel et al., 2012; Wen & Zhang, 2009). However, our study showed that men had a higher chance of social cohesion irrespective of most of them being employed. Similar to our findings, previous studies also describe that older adults, those without a formal education, individuals living alone or in nuclear families, and low income individuals as having lower levels of social cohesion (Pampalon et al., 2007; Rahman & Singh, 2019). Moreover, living in a neighbourhood with low social cohesion increases psychological distress (Erdem, 2019; Rios et al., 2012). A negative association between social cohesion and disability is consistent with other studies that analysed social cohesion and physical activity. In addition, area of residence and neighbourhood affect physical activity (Vancampfort et al., 2018; Yip et al., 2016); higher levels of neighbourhood social cohesion, social participation and trust affects physical activity (Legh-Jones & Moore, 2012), which is essential for reducing disability.\n\nNeighbourhood social cohesion can be considered as a type of social support that is available in the neighbourhood social environment outside of family and friends, which effectively results in the creation and reinforcement of neighbourhood norms (Kim et al., 2014). Neighbourhood social cohesion seems to reduce stress, improve social connections and enforce norms, which ultimately supports a reduction in disability among individuals.\n\nOur study had a few limitations. This is a cross-sectional study, which limits our ability to make causal inferences, like whether disability is pushing individuals to a poorer neighbourhood or whether the poorer neighbourhood is the pertinent factor for disability. Second, since the sample comprised of participants from catchment areas of one state in India, the results cannot be generalised. Third, though the social cohesion scale is extensively used in low and middle income countries (Kulkarni & Shinde, 2015; Fernández-Niño et al., 2019; Ramlagan et al., 2013), it is yet to be culturally validated in our population. Third, most participants were born in villages and nearly half of the population belonged to the low income category. Though the data was taken from respondents residing in that area for more than one year, it does not allow us to adjust for the duration participants were living in their respective neighbourhoods.\n\nNonetheless, our study adds to the literature by presenting evidence on the association of neighbourhood social cohesion and disability conducted in low and middle income countries. Data from our study gives insight to the extent by which the effect of personal and health characteristics on functional ability is mediated by social cohesion. Moreover, data was collected only from the individuals residing in the study area for more than one year, and this indicates a defined neighbourhood.\n\nThe study shows that there is a need to focus on neighbourhood or community level aspects along with individual level aspects to ensure increased quality of life for people with disability. Interventions targeting the entire community can bring about behaviour change in individuals, such as increased physical activity, as we can argue that those who receive positive support for physical activity tend to be more physically active. Our study also calls for a need for interventions offering community-based services, which enhance neighbourhood trust, safety and participation, need to be deployed. Government and policymakers should aim for strategies that enhance neighbourhood cohesion by integrating the physical, public and social health of its people. This will reduce accessibility, availability and affordability issues with respect to disability, as well as increase living standards of people.\n\n\nConclusion\n\nThe present study proposes that there is a significant association between social cohesion and disability, as people with more participation in the community have increased trust and safety and are more likely not to be disabled. Results suggest that improvements in neighbourhood cohesiveness can reduce the level of disability. Future studies can focus on community level interventions that aim to socially integrate people in poorer neighbourhoods, and the instrument used in the present study can be used toassess social cohesion in future studies. A longitudinal study with a more representative sample to help understand the reasons for and strategies to overcome low neighbourhood cohesiveness among people with disability are also essential. Neighbourhood social cohesion seems to reduce stress, improve social connections and enforce norms. Further studies are however needed to study this extensively.\n\n\nData availability\n\nFigshare: Relationship between neighbourhood cohesion and disability: findings from SWADES population-based survey, Kerala, India, https://doi.org/10.6084/m9.figshare.12610607.v3 (Saju et al., 2020c).\n\nFigshare: Relationship between neighbourhood cohesion and disability: findings from SWADES population-based survey, Kerala, India, https://doi.org/10.6084/m9.figshare.12610607.v3 (Saju et al., 2020c).\n\nThis project contains the following extended data:\n\n- Questionnaire consisting of questions used for this study (both English and local language - Malayalam)\n\nSTROBE checklist for ‘Relationship between neighbourhood cohesion and disability: findings from SWADES population-based survey, Kerala, India’, https://doi.org/10.6084/m9.figshare.12610607.v3 (Saju et al., 2020c).\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\nAvlund K, Lund R, Holstein BE, et al.: Social Relations as Determinant of Onset of Disability in Aging. Arch Gerontol Geriatr. 2004; 38(1): 85–99. PubMed Abstract \| Publisher Full Text\n\nErdem Ö: Neighbourhood Structural and Social Factors and Mental Health. Erasmus University Rotterdam, 2019. Reference Source\n\nFernández-Niño JA, Bonilla-Tinoco LJ, Manrique-Espinoza BS, et al.: Neighborhood Features and Depression in Mexican Older Adults: A Longitudinal Analysis Based on the Study on Global AGEing and Adult Health (SAGE), Waves 1 and 2 (2009–2014). Edited by Oliver Gruebner. PLoS One. 2019; 14(7): e0219540. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFiddian-Qasmiyeh E, Loescher G, Long K, et al.: The Oxford Handbook of Refugee and Forced Migration Studies. Oxford University Press. 2014; 785. Publisher Full Text\n\nHyyppä MT, Mäki J: Individual-Level Relationships between Social Capital and Self-Rated Health in a Bilingual Community. Prev Med. 2001; 32(2): 148–55. PubMed Abstract \| Publisher Full Text\n\nKim ES, Hawes AM, Smith J: Perceived Neighbourhood Social Cohesion and Myocardial Infarction. J Epidemiol Community Health. 2014; 68(11): 1020–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKulkarni RS, Shinde RL: Depression and Its Associated Factors in Older Indians: A Study Based on Study of Global Aging and Adult Health (SAGE)– 2007. J Aging Health. 2015; 27(4): 622–49. PubMed Abstract \| Publisher Full Text\n\nLaiglesia J: Perspectives on Global Development 2012. Social Cohesion in a Shifting World? OECD. 2012. Publisher Full Text\n\nLegh-Jones H, Moore S: Network Social Capital, Social Participation, and Physical Inactivity in an Urban Adult Population. Soc Sci Med. 2012; 74(9): 1362–7. PubMed Abstract \| Publisher Full Text\n\nMarti CN, Choi NG: Measuring Social Engagement among Low-Income, Depressed Homebound Older Adults: Validation of the Social Engagement and Activities Questionnaire. Clin Gerontol. 2020; 1–14. PubMed Abstract \| Publisher Full Text\n\nMurray CJL, Barber RM, Foreman KJ, et al.: Global, Regional, and National Disability-Adjusted Life Years (DALYs) for 306 Diseases and Injuries and Healthy Life Expectancy (HALE) for 188 Countries, 1990–2013: Quantifying the Epidemiological Transition. Lancet. 2015; 386(10009): 2145–91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPampalon R, Hamel D, De Koninck M, et al.: Perception of Place and Health: Differences between Neighbourhoods in the Québec City Region. Soc Sci Med. 2007; 65(1): 95–111. PubMed Abstract \| Publisher Full Text\n\nPatel M, Phillips-Caesar E, Boutin-Foster C: Barriers to Lifestyle Behavioral Change in Migrant South Asian Populations. J Immigr Minor Health. 2012; 14(5): 774–85. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRahman MH, Singh A: Disability and Social Cohesion among Older Adults: A Multi-Country Study. Int J Soc Econ. 2019; 46(4): 485–502. Publisher Full Text\n\nRamlagan S, Peltzer K, Phaswana-Mafuya N, et al.: Social Capital and Health among Older Adults in South Africa. BMC Geriatrics. 2013; 13(1): 100. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRios R, Aiken LS, Zautra AJ: Neighborhood Contexts and the Mediating Role of Neighborhood Social Cohesion on Health and Psychological Distress Among Hispanic and Non-Hispanic Residents. Ann Behav Med. 2012; 43(1): 50–61. PubMed Abstract \| Publisher Full Text\n\nSaju MD, Allagh KP, Lorane S, et al.: Prevalence, Awareness, Treatment, and Control of Hypertension and Its Associated Risk Factors: Results from Baseline Survey of SWADES Family Cohort Study. Int J Hypertens. 2020a; 2020: 4964835. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaju MD, Lovakanth N, Shekhar R, et al.: Cohort Profile: Social Well-Being and Determinants of Health Study (SWADES), Kerala, India. BMJ Open. 2020b; 10(3): e032803. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaju MD, Anuja A, Komal PA, et al.: Relationship between neighbourhood cohesion and disability: findings from SWADES population-based survey, Kerala, India. figshare. Dataset. 2020c. http://www.doi.org/10.6084/m9.figshare.12610607.v3\n\nThomas CC, Rathod SD, De Silva MJ, et al.: The 12-Item WHO Disability Assessment Schedule II as an Outcome Measure for Treatment of Common Mental Disorders. Glob Ment Health. 2016; 3: e14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVancampfort D, Stubbs B, Veronese N, et al.: Correlates of Physical Activity among Depressed Older People in Six Low-Income and Middle-Income Countries: A Community-Based Cross-Sectional Study. Int J Geriatr Psychiatry. 2018; 33(2): e314–22. PubMed Abstract \| Publisher Full Text\n\nvan Doorslaer E, O’Donnell O, Rannan-Eliya RP, et al.: Effect of Payments for Health Care on Poverty Estimates in 11 Countries in Asia: An Analysis of Household Survey Data. Lancet. 2006; 368(9544): 1357–64. PubMed Abstract \| Publisher Full Text\n\nWang H, Abajobir AA, Abate KH, et al.: Global, Regional, and National under-5 Mortality, Adult Mortality, Age-Specific Mortality, and Life Expectancy, 1970–2016: A Systematic Analysis for the Global Burden of Disease Study 2016. Lancet. 2017; 390(10100): 1084–1150. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWen M, Zhang X: Contextual Effects of Built and Social Environments of Urban Neighborhoods on Exercise: A Multilevel Study in Chicago. Am J Health Promot. 2009; 23(4): 247–54. PubMed Abstract \| Publisher Full Text\n\nWorld Health Organization: Towards a common language for functioning, disability and health: The International classification of functioning, disability and health. 2002. Reference Source\n\nWorld Health Organization: WHO Global Disability Action Plan, 2014–2021: Better Health for All People with Disability. 2015. Reference Source\n\nWorld Health Organization: World report on disability. 2011. Reference Source\n\nYip C, Sarma S, Wilk P: The Association between Social Cohesion and Physical Activity in Canada: A Multilevel Analysis. SSM Popul Health. 2016; 2: 718–23. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "68096", "date": "31 Jul 2020", "name": "Jed W. Metzger", "expertise": [ "Reviewer Expertise Community organizing", "suicide and homicide research", "international research" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an excellent and important article reflecting on a very high level professional piece of research. Overall, the one critique I have is that it is too short for the type of research - a multivariate study.\nA few specific points to make:\nThe authors are encouraged to spend a little more time developing \"neighborhood\" as an aspect of social cohesion. For instance the males in the study have greater social cohesion- is this due to work outside the home?. For instance perhaps the females spend more of their day as home makers in the community and would have more opportunity for neighborhood social cohesion?\n\nThe authors found 19% of the sample had a disability- is this what the data would expect for Kerala, India?\n\nClearly gender matters- more discussion on this including in promoting social cohesion with those with a disability- it would seem that efforts for males and females may be best if they are gender focused.\n\nAgain this is a very high quality research design. A lot of the strong evidence is in figure two- please expand discussion on that figure.\n\nIn the discussion section, please return to the neighborhood discussion.\n\nThis paper most certainly should be accepted for indexing with these expansions\n\nIs the work 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": [] }, { "id": "68098", "date": "12 Aug 2020", "name": "Debbie Gioia", "expertise": [ "Reviewer Expertise I am only assessing the basic topic of the research and its merits for publication especially as a novel article where no other published research exists." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 important study. I appreciate the authors concisely laid out manuscript and the rationale for the need for this work. I fully appreciate the novel nature of this study and for that reason alone, it is an important component of understanding the effects of neighborhoods on individuals and their health outcomes.\nI confess that I do not have expertise in studies involving large data sets and the types of statistical analysis required to examine the findings from the variable relationships in those data sets, as I am predominately a qualitative researcher. I hope that other reviewers will be able to speak to the methodology chosen.\nAs a mental health researcher, my interest is in the types of reported mental health disorders and community responses, especially during the pandemic. I wonder if there is more specificity in the data set about types of disorders and also the comorbidity with physical health disorders.\nThe source data and conclusions seem very adequate to support the article. Minor edits may still be needed but may also be a result of formatting to the journal's submission portal.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-700
https://f1000research.com/articles/8-1430/v1	14 Aug 19	{ "type": "Method Article", "title": "Development of an informatics system for accelerating biomedical research.", "authors": [ "Vivek Navale", "Michele Ji", "Olga Vovk", "Leonie Misquitta", "Tsega Gebremichael", "Alison Garcia", "Yang Fann", "Matthew McAuliffe", "Michele Ji", "Olga Vovk", "Leonie Misquitta", "Tsega Gebremichael", "Alison Garcia", "Yang Fann", "Matthew McAuliffe" ], "abstract": "Biomedical translational research can benefit from informatics system that support the confidentiality, integrity and accessibility of data. Such systems require functional capabilities for researchers to securely submit data to designated biomedical repositories. Reusability of data is enhanced by the availability functional capabilities that ensure confidentiality, integrity and access of data. A biomedical research system was developed by combining common data element methodology with a service-oriented architecture to support multiple disease focused research programs. Seven service modules are integrated together to provide a collaborative and extensible web-based environment. The modules - Data Dictionary, Account Management, Query Tool, Protocol and Form Research Management System, Meta Study, Repository Manager and globally unique identifier (GUID) facilitate the management of research protocols, submitting and curating data (clinical, imaging, and derived genomics) within the associated data repositories. No personally identifiable information is stored within the repositories. Data is made findable by use of digital object identifiers that are associated with the research studies. Reuse of data is possible by searching through volumes of aggregated research data across multiple studies. The application of common data element(s) methodology for development of content-based repositories leads to increase in data interoperability that can further hypothesis-based biomedical research.", "keywords": [ "Informatics system", "Biomedical repository", "Translational Research", "FAIR" ], "content": "Introduction\n\nTranslational Medicine (TM) seeks to develop new treatments for diseases with insights towards the improvement of global health. To achieve this vision, understanding what and why interventions work, and how they can be scaled to benefit the entire population, depends on successful translational biomedical research and data lifecycle management. The process of TM is time consuming with translational barriers, from basic research to clinical application, bedside to community use, and from community to policy-making decisions. In overcoming the translational barriers, that biomedical informatics platforms reduce the time it takes for basic research to result in clinical applications1. To date, biomedical platforms for translational research have been developed for one or more purposes - (a) management of multi-dimensional heterogeneous data, (b) dissemination of knowledge generated during translational research, (c) testing of analytic approaches for data pipelines, and (d) application of knowledge-based systems and intelligent agents to enable high-throughput hypothesis generation2.\n\nSeveral biomedical informatics applications have been discussed in the literature; for example, Research Electronic Data Capture (REDCap) is a software tool for collecting, storing, creating project specific databases for dissemination of clinical and translational research data3,4. The informatics for integrating Biology and the Bedside (i2b2) system allows researchers to find cohorts of patients that fit specific profiles5. Access to chemical, ‘omics’ and clinical data, with capabilities to investigate genetic and phenotypic relationships for cohorts of patients is supported by tranSMart platform6,7. Analyses of large complex datasets with bioinformatics and image analysis tools, cloud services, application programming interfaces (APIs), and data storage capabilities is supported by the CyVerse infrastructure8,9. Software tools to collect, manage, and share neuroimaging data of different modalities, including magnetic resonance imaging (MRI), magnetoencephalography (MEG), and electroencephalogram (EEG) is available through the Collaborative Informatics and Neuroimaging Suite (COINS)10,11. Also, large scale analysis of biological data can be carried out by web-based platforms12. Within the National Institutes of Health (NIH), the Biomedical Translational Research Information System (BTRIS) has supported researchers to bring together data from the NIH Clinical Center and other institutes and centers13.\n\nHowever, many disease focused research programs have faced data discoverability and integration challenges. For example, traumatic brain injury (TBI) research data was initially collected in different ways and by disparate systems making sharing and reusing of data problematic. Because of the wide variability in systems and databases, many types of TBI injuries were classified as the same class of injury, impeding development of targeted therapies for the disease. To overcome these barriers, the TBI community recommended use of the common data elements (CDE) methodology for the development of the Federal Interagency Traumatic Brain Injury Research (FITBIR)14.\n\nA CDE is defined as a fixed representation of a variable collected within a specified clinical domain, that needs to be interpretable unambiguously in human and machine-computable terms15. It consists of a precisely defined question with a specified format, with a set of permissible values as responses. Typically, CDE development for biomedical disease programs involves multiple steps - identification of a need for a CDE or group of CDEs, stakeholders and expert groups for CDEs selection, iterations and updates to initial development with ongoing input from broader community, with final endorsement of the CDEs by the stakeholder community for its usage and widespread adoption16.\n\nExamples of CDEs use in various programs of clinical research include neuroscience17, rare diseases research18, and management of chronic conditions19. For clinical data lifecycle management, the use of CDEs provides a structured data collection process, which enhances the likelihood for data to be pooled and combined for meta-analyses, modeling, and post-hoc construction of synthetic cohorts for exploratory analyses20. Investigators working to develop protocols for data collection can also consult the NIH Common Data Element Resource Portal for using established CDEs for disease programs21. Also, the feasibility of using common data elements and a data dictionary for the development of the National Database for Autism Research was shown earlier22.\n\nThree key components comprise data dictionaries: data elements, form structures and eForms. A data element has a name, precise definition, and clear permissible values, if applicable. A data element directly relates to a question on a paper, electronic form (eForm) and/or field(s) in a database record. Form structures (FS) serve as the containers for data elements, and the eForms are developed by using FS. The data dictionary provides defined CDEs, as well as unique data elements (UDEs) for specific implementation of the BRICS instance. Reuse of CDEs is significantly encouraged, and in the case of FITBIR’s data dictionary, it incorporates and extends the CDE definitions developed by the National Institute of Neurological Disorders and Stroke (NINDS) CDE Project15.\n\nIn this paper we demonstrate the application of CDE concept for developing a Biomedical Research Informatics Computing System (BRICS), providing functionalities that facilitate electronic submission of research data, validation, curation, and archival storage within program specific data repositories. Use of CDEs enhances data quality and consistency within the repositories that are important for advancing clinical and translational research.\n\n\nMethod\n\nA high level overview of the informatics system architecture is provided in Figure 1. The architecture is defined by the three layers - (a) Presentation Layer, (b) Application Layer, and (c) Data Layer. The Presentation Layer serves as the secure entry point to the BRICS portal. Various open source technologies and libraries, including Java Server Pages (JSP), jQuery, JavaScript libraries (e.g. such as Backbone.js, Asynchronous JavaScript), and XML are used to make web-pages interactive. This layer also includes Web Start applications: Global Unique Identifier (GUID) client, Validation and Upload tools, and Download and Image Submission tools, all of which run on users’ machines.\n\nThe Image Submission Package Creation Tool leverages the 35 plus medical image file readers in the Medical Image Processing Analysis and Visualization (MIPAV) software (v 8.0.2), to make data interoperable, mapping of image header data onto the data elements in imaging form structures for submission to the Data Repository. MIPAV is an open-source software that can be used for image analysis, it is accessible on any Java-compatible platform, including Windows, Mac OS X, and Linux. Over 30 file formats commonly used in medical imaging, including DICOM and NIfTI, and more than ten 3D surface mesh formats are supported by the software23. It also supports multi-scale and multi-dimensional image research from various modalities including microscopy, computerized tomography (CT), positron emission tomography (PET), and MRI. Inclusion of the MIPAV tool with the BRICS provides capabilities for uploading image packages and image analysis, that is not conveniently available on other informatics systems24.\n\nThe Application Layer is responsible for the logic that determines the capabilities of the BRICS modules and tools. Seven service modules within the Application Layer are integrated together to provide a collaborative and extensible web-based environment. These modules are the Data Dictionary (DD), Account Management, Query Tool, Protocol and Form Research Management System (ProFoRMS), Meta Study, Repository Manager and GUID. To communicate and exchange information between the modules, representational state transfer (RESTful) Web services are used.\n\nAdditional information on the various service modules is available from the BRICS site.\n\nThe Data Layer consists of open source databases such as PostgreSQL, Virtuoso databases, file servers, and data persistence frameworks. The Virtuoso database is used to store the data accessed by the Query Tool and to store CDE metadata in the Data Dictionary data. The Repository module uses the PostgreSQL database to store and retrieve data. Also utilized are open-source libraries such as Hibernate and Apache Jena for storing and retrieve data from databases. The data layer is supported by the physical infrastructure located within the National Institutes of Health, and is certified at the Federal Information Security Modernization Act (FISMA) Moderate level25, conforming to additional USA federal information standards26,27.\n\nTo de-identify data, researcher’s use the GUID tool (shown as a client in Figure 1) to assign a unique identifier for each study participant. The GUID is a random alphanumeric unique subject identifier (not generated from personally identifiable information (PII). The PII fields that can be used as part of the hashing process include complete legal given (first) name of subject at birth, middle name (if available), complete legal family (last) name of subject at birth, day of birth, month of birth, year of birth, name of city/municipality in which subject was born, and country of birth. The PII data is not sent to the GUID server but rather one-way encrypted hash codes are created and sent from the GUID client to the server (represented as a service module, Figure 1), allowing the PII to reside only on the researcher’s site. A random number for each of the research participant is generated by the server and is returned to the researcher. In addition, the GUID server can be configured to support multi-center clinical trials and investigations that enroll research participants across various programs.\n\nResearchers are responsible for most of the data submission activities, which includes study FS approval, eForms review, curation, mapping of data elements, and providing associated study documentation.\n\nTwo routes of data submission are available for researchers to make data findable. One approach is by using the ProFoRMS tool (Figure 2, stage 1) for clinical research work, scheduling subject visits, collecting data, adding new data, modifying previously collected data entries, and correcting discrepancies that are tracked and maintained in audit logs. The other mode is by using a generic data collection system (e.g. RedCap), validating with the BRICS data dictionaries and uploading the extracted data into the repository module (Figure 2, stage 2). Both routes of data submission validate the submitted data using specific range and values from the data dictionaries for a BRICS instance.\n\nThe Validation Tool supports the data repository and ProFoRMs modules, by using CDEs with defined range and value metrics for data quality checks, to make data reusable. Once the data has been validated and uploaded via the submission upload tool, data is stored in its raw form within the repository module in a database that can be accessed by the Query Tool (Figure 2, stage 3).\n\nUser support is provided for data stewardship activities that include training and assistance to authorized users, for CDE implementation, data validation and submission to the repositories. Access is controlled by a Data Access Committee (DAC) that reviews studies for relevance to a specified BRICS instance (defined by the biomedical program). In addition, access to the system is role based and specific permissions are associated with roles such as PI, data manager, and data submitter.\n\nDuring packaging of data, GUIDs are assigned to research subjects (patients), using the GUID client with the users responsible for storing PII data locally within their institutional systems. Data curation is carried out by identifying the available standard forms and CDEs in the Data Dictionary. In the event no corresponding CDEs are available, then the user can define the data elements and obtain approval during the submission process.\n\nThe data Repository module serves as a central hub, providing functionality for defining and managing study information and storing the research data associated with each study (Figure 2, stage 3). Authorized investigators can submit data to a BRICS instance, organize one or many datasets into a single entity called a Study. In general terms, a ‘Study’ is a container for the data to be submitted, allowing an investigator to describe, in detail, any data collected, and the methods used to collect the data, which makes data accessible. By using the repository user interface, researchers can generate digital object identifiers (DOIs) for a study, which can be referenced in research articles.\n\nThe repository module provides download statistics for specific studies, enabling the investigator to obtain information on their respective data that has been downloaded for other research activities, and overall increase data sharing and collaboration for additional research goals. Depending on the research studies, BRICS based repositories can host high throughput gene expression, RNA-Seq, SNPs, and sequence variation data sets (Figure 2, stage 3).\n\nBy default, the system assigns the sharing preference as ‘private’ where only users to that specific study can access the data. When the data is in the private state, the PI has the option to share data with specific collaborators (preferential sharing). After a certain period (defined by the data sharing policy for each BRICS instance), the data enters a new ‘shared’ state, which is accessibleto the approved users.\n\nRaw data is available for querying within 24 hours of data submission. For the data to be available via the Query Tool module, the raw data is processed through the ‘NextGen Connect’ tool (integrated interface engine) and Resource Description Framework (RDF) data interchange tool (Figure 2, stage 4). Shared data is available to all system users (approved by DAC) to search, filter, and download via the Query Tool functionality. The Query Tool offers three types of functionalities - (a) querying and filtering data, (b) data package downloads based on query, and (c) data package to the Meta Study module.\n\nThe Meta Study module is used for meta-analysis of the data as well as a collaboration tool between scientific groups. A Meta Study contains findings from studies that can be aggregated by researchers to conduct additional analysis. The Query tool can also support the statistical computing language R as well as structured visualization of data (Figure 2, stage 4).\n\n\nResult\n\nThe Query Tool (QT) enables users to browse studies, forms and CDEs, to select clinical data, use filters, and to sort and combine records. Using the GUID and a standard vocabulary via CDEs in forms, the QT provides an efficient means to reuse data by searching through volumes of aggregated research data across studies, find the right datasets to download and perform offline analysis using additional tools (e.g. SAS, SPSS, etc.). The statistical ‘R-box’ tool, integrated with BRICS, has been incorporated in the QT, to support analysis without having to download data.\n\nThe QT has several ways to search for data. By default, the user is presented with all studies in the data repository that have data submitted against them. Users can use the QT to search for desired data by searching by study, or across studies by form or an individual data element (Figure 3a).\n\nExample from the Parkinson’s Disease Biomarker Program BRICS instance.\n\nEach column of data in a QT result represents a well-defined element in the Data Dictionary. Users can refine results by selecting from the list of allowed element permissible values, like male or female, or move sliders to select a range of numeric values, like age or outcome scores (shown in Figure 3b).\n\nIn addition to providing tools to aid data discovery, the QT supports interactive features that facilitate analysis and practical use of the data through attribute-based filtering capabilities, based on the data element type.\n\nVarious datasets (e.g. clinical, cognitive, demographic) are available within the repositories that are integrated to the BRICS instances. Data can be shared in CSV file format for download, and/or stored in the Meta Study module for further analysis, research, and reference.\n\nThe Parkinson’s Disease Biomarker Program (PDBP) signifies the importance of Parkinson’s disease biomarker discovery process, which requires data replication and validation prior to clinical trial use28. Making both research data and workflow process findable, accessible, interoperable, and reusable was an important design consideration during the development of the PDBP system. The system consists of two major components - (a) Drupal-based portal, and (b) the PDBP Data Management Resource (DMR). The portal is publicly accessible to users for obtaining policy, stakeholders, individual PI, and specific study information, including summary data, and news (see PDBP site). The PDBP DMR is comprised of the previously discussed BRICS modules (shown in Figure 2) and incorporated the Parkinson’s disease CDEs into its Data Dictionary29. Use of CDEs results in making data FAIR by harmonization of clinical, imaging, genomics, laboratory, and biospecimen data. The CDEs are easily accessible from multiple open resources - the PDBP data dictionary30, the NINDS CDE project15, and the NIH CDE repository21. The DMR is securely managed with capabilities for account verification, GUID generation, data submission, validation, workflows, access, and biospecimen data management. A GUID is generated for each subject on their initial visit and is attached to the deidentified data. The GUID makes data reusable by enabling the aggregation of all research data (clinical, imaging, genomic, and biomarker) for a specific subject, both within a single study and across many PDBP studies.\n\nThe ProFoRMS module (shown in Figure 2) is used to schedule Parkinson’s Disease subject visits and capture data (including the GUID) via a web-based assessment form tool. It provides capabilities for real time data entry and automatic data harmonization and ensures data quality assurance prior to storage within the PDBP repository. Each of the questions in the PDBP DMR assessment form is associated to a CDE that supports reusability and interoperability of PDB data28,31. The ProFoRMS also provides automatic assignment of specific forms to individualized cohorts based on protocol design, and quality assessment of data prior to uploading to the PDBP Data Repository.\n\nThe authorized PDBP users can use the QT for accessing data across studies and aggregate data based on assessment forms and CDEs, allowing for the linkage of biosample data to demographics data. More complex queries can be created by linking clinical data from ProFoRMS with imaging data from the MIPAV module and with corresponding biospecimens/biosamples.\n\nData can be downloaded directly from the PDBP data repository and/or from the Query Tool to be analyzed by researchers using their preferred tools. Because the DMR database contains only de-identified data, all data uploaded to the DMR can be shared with the scientific community. Use of standard operating procedures has resulted in harmonization of biospecimens/biosamples with the DMR Biosample Order Manager tool, which enables linking clinical and biorepository data32. The PDBP data, queries and other metadata described for the research can be loaded into the Meta Study module and through the Meta Study user interface researchers can generate DOIs that can be referenced in research articles.\n\nThe initial deployment of BRICS was to support the U.S. Department of Defense's (DoD) and the National Institute of Neurological Disorders and Strokes (NINDS), FITBIR project. The core functionalities for FITBIR were reusable for developing PDBP, as well other biomedical programs.\n\nA few highlights of the data repositories resulting from the implementation of BRICS instance for the biomedical programs are provided below -\n\nFederal Interagency Traumatic Brain Injury Research (FITBIR). is a BRICS instance developed to advance comparative effectiveness research in support of improved diagnosis and treatment for those who have sustained a TBI33. The FITBIR repository stores data provided by TBI researchers and has accepted high quality research data from several studies, regardless of funding source and location. The DoD and NINDS provides funding for TBI human subject studies (both retrospective and prospective) and have required the research grantees to upload their clinical, imaging, and genomic data to FITBIR. As of 2018, there are 157 studies in FITBIR, spanning nearly hundred PIs, dozens of universities and research systems, the DoD, and the NIH. Data on 69,208 subjects, including more than 82,000 clinical image 3D data sets that are part of the repository. Currently, there are a total of 1,857,926 records in FITBIR. Data provided to FITBIR for broad research access are expected to be made available to all users within six months after the award period ends.\n\nParkinson’s Disease Biomarkers Program Data Management Resource (PDBP DMR). is a BRICS instance developed to support new and existing research and is a resource for promoting biomarker discovery for Parkinson's disease funded by NINDS, NIH. At the center of the PDBP effort is its DMR. The PDBP DMR uses a system of standardized data elements and definitions, which makes it easy for researchers to compare data to previous studies, access images and other information, and order biosamples for their own research. PDBP’s needs have accelerated BRICS system development, such as enhancements to the ProFoRMS data capture module, also with an investment into a BRICS plug-in for managing biosamples. The PDBP DMR now contains over 1,500 enrolled subjects, 1,415 of whom have biorepository samples. Also, PDBP has currently a total of 55,400 records.\n\neyeGENE. has a BRICS instance to support the National Ophthalmic Disease Genotyping and Phenotyping Network34. It is a research venture created by the National Eye Institute (NEI) to advance studies of eye diseases and their genetic causes, by giving researchers access to DNA samples and clinical information. Data stored in eyeGENE is cross-mapped to Logical Observation Identifiers Names and Codes terminology (LOINC) interoperability data standards35. Currently, eyeGene has 146,024 records with 6,400 enrolled subjects.\n\nInformatics Core of Center for Neuroscience and Regenerative Medicine (CNRM) has a BRICS instance to support the CNRM medical research program with collaborative interactions between the U.S. DoD, NIH, and the Walter Reed National Military Medical Center. The Informatics Core provides services such as electronic data capture and reporting for clinical protocols, participation in national TBI research and data repository community, integration of CNRM technology requirements, and maintenance of a CNRM central data repository36. In addition, the Informatics Core has played an important role in the development of multiple BRICS modules used by FITBIR.\n\nCommon Data Repository for Nursing Science (cdRNS) has a BRICS instance to support the National Institute of Nursing Research (NINR) mission - to promote and improve the health of individuals, families, and communities37. To achieve this mission, NINR supports and conducts clinical and basic research and research training on health and illness. This research spans and integrates the behavioral and biological sciences, and that develops the scientific basis for clinical practice38. The NINR is a leading supporter of clinical studies in symptom science and self-management research. To harmonize data collected from clinical studies, NINR is spearheading an effort to develop CDEs in nursing science. Currently, there are 1,358 records in the cdRNS instance of BRICS.\n\nThe Rare Diseases Registry. has a BRICS instance for the Rare Diseases Registry (RaDaR) program of the National Center for Advancing Translational Sciences (NCATS). It is designed to advance research for rare diseases39. Because many rare diseases share biological pathways, analyses across diseases can speed the development of new therapeutics. The goal is to build a Web-based resource that integrates, secures, and stores de-identified patient information from many different registries for rare diseases, all in one place.\n\n\nDiscussion\n\nThe informatics system utilizes the Open Archival Information System (OAIS) model for preserving information for a designated community (group of potential consumers and multiple stakeholders). The implementation of the model highlights the importance of developing Submission, Archival, and Dissemination Information Packages (Figure 2, SIPs, AIPs and DIPs) for longer term data preservation and reuse40. The primary producers of the data for the informatics system are the researchers and staff associated with each of the biomedical programs. Clinical data SIPs are produced for each of the instances by using eCRFs and imaging data SIPs are produced by the Image Submission tool. The CDEs and data dictionaries for the various BRICS instances support the development of archival information packages (AIPs), which are stored in distinct data repositories identified by the biomedical research programs41. The portability of the informatics software is also possible by recent deployment to the National Trauma Research (NTR) data repository development work42. In contrast to most of the centrally managed repositories within the NIH, the informatics software hosted for NTR is within a secure Amazon Web Services cloud platform. Deploying in the cloud environment enhances data access, sharing, and reuse of biomedical research data at larger scale43.\n\nThe FAIR (Findable (F), Accessible (A), Interoperable (I), and Reusable (R)) principles state that stewardship of digital data should promote discoverability and reuse of digital objects, which includes data, metadata, software and workflows44. In addition, the principles posit that data and metadata should be accompanied by persistent identifiers (PIDs), indexed in a searchable resource, retrievable by their identifiers, and use vocabularies that meet domain relevant community standards. The principles serve as guidelines for developing systems that can improve data discovery and reuse. In Table 1, we have correlated the various BRICS functional components, which contribute towards making data FAIR for biomedical research programs.\n\nThe FAIR principles listed in the table are from the cited reference 44.\n\nUnique identification that is machine-resolvable with a commitment to persistence is fundamental for providing access to data and metadata45. In the context of the informatics system discussed here, GUID does not imply findability on the web, however, it purports findability of research participant data within a BRICS instance. Authorized researchers can use GUID to link together all submitted information for a single participant, even if data was collected at different locations and/or for different purpose(s).\n\nSeveral identifier schemes (e.g. DOI, Handle system, Identifiers.org, Uniform Resource Identifiers) vary in their characteristics46. A fundamental difference in an identifier scheme can be in the management (centrally or locally) of the resolver. For example, in the DOI scheme, a dereferencing service (e.g. Datacite or CrossRef) serves as a resolver that redirects the identifier to the actual content and the metadata.\n\nThe DOIs generated by BRICS is through the Interagency Data ID Service (IAD), which is operated by the U.S. Department of Energy Office of Scientific and Technical Information (OSTI). The IAD service acts as a bridge to DataCite, which is one of the major registries of DOIs. The DOIs are assigned to individual research studies and are findable within the established repositories, available also from open sites with core metadata supported via Data Tag Suite (DATS) 2.247.\n\nThe availability of an automated validation tool with the informatics system makes CDE findable in the Data Dictionary and ensures for data quality and consistency. The system provides for an automated means of mapping CDEs to other informatic systems data dictionaries, e.g. CDISC48. CDEs are made available through public websites (e.g. National Library of Medicine (NLM), NINDS CDE project, CDISC, etc.) to make data interoperable. Usability of data is enhanced by the adoption of standard imaging formats (e.g. DICOM, NIFTI, etc.). The informatics system also supports data discoverability across multiple repositories through the application of the biomedical and healthCAre Data Discovery Index Ecosystem (bioCADDIE)49.\n\n\nConclusion\n\nData confidentiality, integrity and accessibility are essential elements of responsible biomedical research data management. Community-wide data sharing requires development and application of informatics systems that promote collaboration and sustain data integrity of research studies within a secure environment. The informatics system presented above enables researchers to efficiently collect, validate, harmonize, and analyze research datasets for various biomedical programs. Integration of the CDE methodology with the informatics design results in sustainable digital biomedical repositories that ensure higher data quality. Aggregating data across projects, regardless of location and data collection time can define study populations of choice, for exploring new hypotheses based-research.\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\nSource code available from: https://github.com/brics-dev/brics\n\nArchived source code at time of publication: http://doi.org/10.5281/zenodo.335572750\n\nLicense: Other (open). Full license agreement is available from GitHub (https://github.com/brics-dev/brics/blob/master/License.txt)", "appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgment\n\nThe authors thank Mr. Denis von Kaeppler, Center for Information Technology, National Institutes of Health for helpful discussions and suggestions during the preparation of the manuscript, Ms. Abigail McAuliffe and Mr. William Gandler, Center for Information Technology, National Institutes of Health for editing the manuscript.\n\nThe opinions expressed in the paper are those of the authors and do not necessarily reflect the opinions of the National Institutes of Health.\n\n\nReferences\n\nSarkar IN: Biomedical informatics and translational medicine. J Transl Med. 2010; 8: 22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPayne PR: Chapter 1: Biomedical knowledge integration. PLoS Comput Biol. 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Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3355727" }	[ { "id": "56129", "date": "18 Nov 2019", "name": "Hyeoneui Kim", "expertise": [ "Reviewer Expertise biomedical informatics", "standardized data representation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 introduces the Biomedical Research Informatics Computing System (BRICS), a comprehensive platform that supports researchers collect, store, analyze, and securely share research data. The underlying principle that motivated and enabled the implementation of this system is the FAIR (Findable, Accessible, Interoperable, and Reusable) principle for biomedical data. The authors provided clear and highly informative descriptions of the architecture and the approaches to implementing the key functional components. The related initiatives and programs that aim at improving data use and reuse and the gaps found in them provide a convincing context for BRICS development. The figures presented in the paper adequately describe the structure, functions, and workflows of BRICS. The research programs and initiatives that already utilize BRICS introduced in the paper are strong evidence that supports the BRICS approach. All in all, this is a well-written, very informative paper. The changes suggested below could help strengthen this paper even more:\n\nThe conventional section structure that includes methods and results might not fit well with this paper. If F1000Research allows some flexibility in the manuscript structure, it will help readers follow the progress of the content by changing the Method section to something like BRICS Functionalities and Components (or something along this line) and Result to BRICS Instance (or use cases). Also, the query module explained in the result section can be included in the BRICS functionalities and components section.\n\nA brief description of how the study level metadata (e.g., sample size, study design, study location, etc.) are captured would be helpful as study-level metadata are among the most frequently used parameters for data/dataset search.\n\nAccessing a broader scope of data is one of the main motivations that researchers would adopt this type of data platform. Therefore, although it is not related to the technological development of BRICS, introducing more information on the data sharing policies (i.e., data use agreement and DAC's responsibilities) would be beneficial.\n\nPlease correct minor errors, such as typos and introducing BRICS first without fully spelling out the acronym.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "5692", "date": "13 Jul 2020", "name": "Vivek Navale", "role": "Author Response", "response": "1. Thank you for the suggestions. To improve clarity, we have redefined method section as BRICS System Design and Architecture, followed by two sections - Data Submission and Processing, Sharing and Access sections. We have integrated the Query Tool description within the section the Data Access section. 2. Under the data submission and processing section, we have added information on study level metadata, that is entered manually through a graphical user interface, when a BRICS instance is used. The examples of Metadata fields include title, organization, PI, data, funding source and ID’s, study type(s), and keywords that enable users to search for detailed information (e.g. clinical trial Grant ID(s), start and end dates for grants, therapeutic agents, sample size, publications, and forms used).We have indicated that each of the BRICS instance exposes metadata and summary consistent with their respective program goals. We have provided an example, FITBIR provides a metadata visualization tool that graphically supports searching study identification (shown here https://fitbir.nih.gov/visualization).3.Thank you for the suggestion. We have added information in the Data Sharing and Access section indicating that each instance of BRICS supports the data sharing policies consistent with their respective program. Research data is maintained in a private state until a year after the grant end date, and after that time, data is moved to a shared state where all users with approval from DAC can have access to the data. The DAC is comprised of government program officials responsible for each of the BRICS instances, who evaluate the data access requests and approve or disapprove the request. A detailed information for each of the BRICS instances can be gleaned from the site information (web site links) provided under the BRICS instance section." } ] }, { "id": "55222", "date": "03 Dec 2019", "name": "Timothy W. Clark", "expertise": [ "Reviewer Expertise Biomedical informatics. Semantic technologies. Cloud computing frameworks. Neuroscience. Data science." ], "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 article which one eventually learns is about the BRICS system, developed for NINDS by software consultants at General Dynamics and Sapient. It is poorly organized, and seems to take little care to actually explain the system's motivation and use in a coherent fashion understandable to the reader.\n\nThe authors begin with an overgeneralized and basically non-informative abstract and introduction which make a number of broad statements about translational research and its requirements for enabling informatics. But one does not discover the actual real focused topic of the article until far down in the text.\nOne does not learn, for example, until page 8, the specific motivation of the system:\n\"The initial deployment of BRICS was to support the U.S. Department of Defense’s (DoD) and the National Institute of Neurological Disorders and Strokes (NINDS), FITBIR project. The core functionalities for FITBIR were reusable for developing PDBP, as well other biomedical programs.\"\n\nObviously there has been a major software development effort conducted by contractors funded and managed by NINDS, and this article is attempting to report on what was accomplished. The implementation is not interesting in itself, using mostly 20-year-old technology. However, the scale of the software effort and its importance to researchers working on certain NIH funded projects, would seem to require something far better as a report out, whether or not it was accepted by a peer-reviewed journal.\n\nCrucial rationales for technical decisions made, and contexts in which the system is used, are omitted or poorly motivated, or described inaccurately. For example, we learn on page 4 that:\n\"The Data Layer consists of open source databases such as PostgreSQL, Virtuoso databases, file servers, and data persistence frameworks.\"\nVirtuoso is not an open-source database. It is an enterprise-class RDF graph store. Nowhere up to now has the need for an RDF graph store been explained, and it is not touched on again until page 6, where we learn that:\n\"The raw data is processed through the ‘NextGen Connect’ tool (integrated interface engine) and Resource Description Framework (RDF) data interchange tool.\"\n\nAnd that is all we ever learn about the use of RDF or any related semantic technologies in this system. Are there OWL Ontologies involved? Why was the decision made to use them and which ones were selected? Why is Virtuoso used in conjunction with PostgreSQL relational store? What makes this combination necessary? We hear nothing of this.\n\nThis reviewer prepared a line-by-line discussion of the text which can be found here. Suffice it to say that there are many significant issues with this article - issues of poor exposition, lack of required detail or context, imprecision, or simply misleading statements.\nAnother example:\n\"The Meta Study module is used for meta-analysis of the data as well as a collaboration tool between scientific groups. \"\n\nThat sentence is all we ever hear of the ability to perform meta-analysis, or any of the challenges it poses.\n\nOr this:\n\"Deploying in the cloud environment enhances data access, sharing, and reuse of biomedical research data at larger scale\".\n\nIn fact the reason to deploy things in the cloud is for rapid horizontal and vertical scaling. lt has nothing to do with data sharing and reuse. The authors cite an article by one of them (Navale & Bourne 2018), to back up their incorrect claim - but that article directly contradicts this claim.\n\nI strongly recommend to the authors that they engage a high-quality technical writing firm competent in bioinformatics to revise the text, paying close attention to precision, providing needed context for technical choices, context of usage and overall motivation of the system, and in general, thoughtful informative exposition.\n\nIs the rationale for developing the new method (or application) clearly explained? No\n\nIs the description of the method technically sound? Partly\n\nAre sufficient details provided to allow replication of the method development and its use by others? No\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? No", "responses": [ { "c_id": "5693", "date": "13 Jul 2020", "name": "Vivek Navale", "role": "Author Response", "response": "1. Thank you for the comments. We have revised the manuscript extensively and addressed the detailed comments that you provided us. The organization of the manuscript has been made easier for readers to follow, appropriate headings have been contextualized throughout the paper. The introduction has been refocused, motivation highlighted and the specific points that were brought to our attention have been addressed in the manuscript.2. We have revised the abstract and focused on the BRICS functional components, services deployed for research data life cycle management and demonstrated the application to various biomedical research programs. The common data element concept has been contextualized and the significance to this work has been highlighted.3. Thank you for the comments on the BRICS Architecture section. We have made major revisions to this section to explain the design choices during the development work, lessons learned and accurately depicted the current architecture in Figure 1.4. We have clarified in the Data Submission section that institutional grants (e.g. DOD, NIH) support disease specific research and mandate data to be submitted to a specific BRICS instance. 5. We have revised the section to state that after data has been validated and uploaded to the repository, an original copy of the user submitted data (raw data) is maintained in the repository that can be accessed by the Query Tool.6. Thank you for your suggestions. We have revised the section to clarify that data quality and consistency of submissions is enhanced by validation, using domain specific Data Dictionaries.7. We agree that the biocaddie.org is not available, the project has been discontinued, hence access to BRICS repositories via bioCADDIE will not be possible. Therefore, we have removed the statements from the revised manuscript.8. We have revised the section on BRICS instances and specified that the National Trauma Research Repository utilizes Cloud Computing for providing infrastructure as-a-service." } ] } ]	1	https://f1000research.com/articles/8-1430
https://f1000research.com/articles/8-1911/v1	12 Nov 19	{ "type": "Research Article", "title": "Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour", "authors": [ "Jamie R. Bhagwan", "Emma Collins", "Diogo Mosqueira", "Mine Bakar", "Benjamin B. Johnson", "Alexander Thompson", "James G.W. Smith", "Chris Denning", "Emma Collins", "Diogo Mosqueira", "Mine Bakar", "Benjamin B. Johnson", "Alexander Thompson", "James G.W. Smith" ], "abstract": "Background: Diseases such as hypertrophic cardiomyopathy (HCM) can lead to severe outcomes including sudden death. The generation of human induced pluripotent stem cell (hiPSC) reporter lines can be useful for disease modelling and drug screening by providing physiologically relevant in vitro models of disease. The AAVS1 locus is cited as a safe harbour that is permissive for stable transgene expression, and hence is favoured for creating gene targeted reporter lines. Methods: We generated hiPSC reporters using a plasmid-based CRISPR/Cas9 nickase strategy. The first intron of PPP1R12C, the AAVS1 locus, was targeted with constructs expressing a genetically encoded calcium indicator (R-GECO1.0) or HOXA9-T2A-mScarlet reporter under the control of a pCAG or inducible pTRE promoter, respectively. Transgene expression was compared between clones before, during and/or after directed differentiation to mesodermal lineages. Results: Successful targeting to AAVS1 was confirmed by PCR and sequencing. Of 24 hiPSC clones targeted with pCAG-R-GECO1.0, only 20 expressed the transgene and in these, the percentage of positive cells ranged from 0% to 99.5%. Differentiation of a subset of clones produced cardiomyocytes, wherein the percentage of cells positive for R-GECO1.0 ranged from 2.1% to 93.1%. In the highest expressing R-GECO1.0 clones, transgene silencing occurred during cardiomyocyte differentiation causing a decrease in expression from 98.93% to 1.3%. In HOXA9-T2A-mScarlet hiPSC reporter lines directed towards mesoderm lineages, doxycycline induced a peak in transgene expression after two days but this reduced by up to ten-thousand-fold over the next 8-10 days. Nevertheless, for R-GECO1.0 lines differentiated into cardiomyocytes, transgene expression was rescued by continuous puromycin drug selection, which allowed the Ca2+ responses associated with HCM to be investigated in vitro using single cell analysis. Conclusions: Targeted knock-ins to AAVS1 can be used to create reporter lines but variability between clones and transgene silencing requires careful attention by researchers seeking robust reporter gene expression.", "keywords": [ "Human induced pluripotent stem cells", "CRISPR/Cas9", "stem-cell derived cardiomyocytes", "stem-cell derived haematopoietic cells", "AAVS1 safe harbour", "gene targeting", "silencing" ], "content": "Introduction\n\nA key consideration for targeted gene delivery in human induced pluripotent stem cells (hiPSCs) is the genomic location at which to insert the exogenous DNA sequence to maximise transgene expression and limit disruption of critical endogenous genes and their function. To this end, a number of chromosomal locations that are amenable to integration have been exploited. These regions of the genome are commonly referred to as safe harbour loci, and often share some common properties such as limited disruption to endogenous genes, low proximity to oncogenes and a chromatin structure that is not prone to epigenetic silencing1,2.\n\nExamples of previously utilised genomic safe harbour loci include the chemokine (C-C motif) receptor 5 (CCR5) gene3,4, the human orthologue of the mouse Rosa26 locus (hROSA26)5, and a region within intron 2 of the Citrate Lyase Beta-Like (CLYBL) gene6.\n\nThe AAVS1 locus is an area of chromosome 19 (position 19q13.42) that has been found to be a common integration site for exogenous DNA delivered to cultured cells with adeno-associated virus (AAV)7,8. Integration into this site is associated with only limited disruption of endogenous genes. The phosphatase 1 regulatory subunit 12C (PPP1R12C) gene codes for a protein with a poorly defined function, and its first intron is disrupted by integration into the AAVS1 site, with no observed deleterious consequences in targeted human pluripotent stem cells (hPSCs)2,9. DNA sequences inserted at this location are supposedly protected by endogenous insulator regions10. These insulators are considered to contribute to maintaining an open chromatin conformation at the AAVS1 locus, reducing the likelihood of transgene silencing compared to other safe harbour loci such as CCR54,11. However, some reports of DNA methylation dampening transgene expression in hPSC-derived hepatocytes raise questions on whether a ‘perfect’ safe harbour locus exists12. Despite this, AAVS1 has remained popular for gene targeting13–16.\n\nWe sought to utilise CRISPR/Cas9 nickase to target the AAVS1 locus in hiPSCs and introduce a genetically encoded calcium indicator, R-GECO1.0, to enable live Ca2+ imaging in hiPSC-derived cardiomyocytes (hiPSC-CMs)17,18. This was performed in genome engineered isogenic hiPSC lines we previously described to model the condition, hypertrophic cardiomyopathy (HCM). These included a trio of lines harbouring a c.MYH7C9123T mutation19 and a duo harbouring a c.ACTC1G301A mutation20.\n\nIn addition, CRISPR Cas9 targeting of the AAVS1 locus was used to target a doxycycline-inducible HOXA9-T2A-mScarlet cassette into hiPSCs with the aim of modulating HOXA9 during haematopoietic differentiation.\n\nWe found that, far from being a safe harbour locus, AAVS1 integration associated with transgene expression that varied between clones and/or was silenced during directed differentiation towards both haematopoietic cells and cardiomyocytes. This suggests that silencing at the AAVS1 locus is not limited to the endoderm lineage as previously described12. Nevertheless, by altering our methods from bulk population analysis to single cell confocal laser line scan microscopy, we used the hiPSC-CMs expressing R-GECO1.0 to investigate the impact of HCM mutations on Ca2+ transients. Abnormalities were found in both HCM-associated mutations c.MYH7C9123T and c.ACTC1G301A, and this phenotype was successfully rescued with drug treatment. This demonstrates an in vitro alternative to some aspects of drug testing on animal models of HCM. Finally, we conclude that the AAVS1 locus cannot be considered a true safe harbour. Researchers seeking to target this locus should check clones for transgene expression status both in hiPSCs and in differentiated progeny.\n\n\nMethods\n\nInformed patient consent was obtained for all patient-derived hiPSC samples to be used for research purposes. Isolation and use of patient fibroblasts was approved by the Nottingham Research Ethics Committee (License 09/H0408/74), and sample collections are registered with the UK Clinical Research Network under project 8164.\n\nAll cell culture experiments were performed in a type II Biological Safety Cabinet, and cells were incubated in a humidified incubator at 37°C and 5% CO2. hiPSCs were routinely maintained in E8 medium on 1:100 Matrigel (Corning #356235) coated plastic ware (Nunc). Cells were passaged every three days by washing once with Ca2+/ Mg2+-free Phosphate Buffer Saline (PBS, Gibco #14190-094), followed by incubation with TrypLE for four minutes. Subsequently, hiPSC were resuspended in E8 supplemented with 10 μM Y-27632 (ROCKi, Tocris Bioscience #1254/10) and seeded into new Matrigel-coated flasks at approximately 20000 cells/cm2. Medium was changed every day.\n\nhiPSC differentiation to cardiomyocytes was performed as previously described19,20. Briefly, culture vessels were seeded at approximately 20–40 thousand cells / cm2, followed by a Matrigel™ overlay step two days later, supplemented with 1 ng/ml BMP4 [R&D #314-BP-050]. 16 hours later, medium was changed to StemPro™34- Serum Free Medium [SP34, Gibco #10639011], supplemented with 8 ng/ml Activin A (ActA, LifeTechnologies #PHC9564) and 10 ng/ml BMP4. After 48 hours medium was changed to RPMI B27 without insulin (LifeTechnologies #A1895601), with 10 μM KY0211 (R&D #4731) and 10 μM XAV939 (R&D #3748). 48 hours later, medium was changed to RPMI B27 (LifeTechnologies #0080085-SA) with 10 μM KY0211 and 10 μM XAV939. Thereafter, medium was changed every 2–3 days with fresh RPMI B27 until day 15 of differentiation, when hiPSC-CMs were dissociated using collagenase21, re-plated, and kept in RPMI B27 for another week until phenotypic assays were performed.\n\nhiPSC differentiation to haematopoietic cells was performed by passaging hiPSCs using Gentle Cell Dissociation Reagent (GCDR; Stem Cell Technologies #07174) and Corning cell scrapers (Sigma #CLS3008) when colonies had compacted and wells were 70–80% confluent. Differentiation was performed using STEMdiff™ Haematopoietic Kit according to the supplied protocol (Stem Cell Technologies #05310). Briefly, hiPSCs were dissociated as cell aggregates with GCDR for 7–10 minutes at room temperature, followed by scraping. Cell aggregates (50–200 µm in diameter) were seeded at different ratio densities in E8 media. The following day, only wells that contained 16–40 colonies >50 µm in diameter were selected to continue with differentiation and media was changed to Media A (day 0 of differentiation). On day two, a half Media A change was performed. On day three, media was changed to Media B and half Media B changes were performed on days five, seven and 10. On day 12, suspension cells were harvested for downstream analysis.\n\nIn order to target the AAVS1 locus in hiPSCs, a targeting vector was constructed containing either the CAG-R-GECO1.0-IRES-Puro cassette17 or the doxycycline-inducible HOXA9-T2A-mScarlet-CAG-G418 cassette flanked on each side with 1 kb of homology to the AAVS1 locus22. 1 µg of AAVS1 targeting vector was transfected into 1 × 106 hiPSCs, with 500 ng of each AAVS1 guide RNA pU6 vector and 1 µg of hCas9 D10A nickase plasmid using an Amaxa 4D system (Lonza) according to the manufacturer’s instruction. 24 hours after transfection, the medium was supplemented with 0.3 µg/ml puromycin (Life Technologies #A1113802) or 50 µg/ml Geneticin™ (Life Technologies #10131027) depending on the drug selection cassette for positive selection of clones up to 10 days post-transfection. Drug-resistant clones were then isolated using 0.5 mM EDTA and expanded. Clones were then genotyped using polymerase chain reaction (PCR) on genomic DNA using Phusion® polymerase (NEB Cat# M0530S) and the primers given in Table 1. PCR cycle parameters were 95°C for 2 minutes, 60–64°C for 30 seconds and 72°C for 60 seconds, with a final elongation step of 72°C for 10 minutes.\n\nDissociated hPSC-CMs or hPSCs were cultured in vitronectin- or MT-coated 96-well plates (CellCarrier, Perkin Elmer #6005550), respectively, at approximately 50K cells/cm2 as described above. Cells were washed with PBS and fixed in 4% Paraformaldehyde (PFA, Sigma) at room temperature (RT) for 15 minutes. Afterwards, cells were washed in 0.1% Tween-20 (Fisher Scientific) in PBS, permeabilized with 0.1% Triton-X (Sigma) in PBS for 15 min at RT, and incubated with 4% goat serum (Sigma) in PBS (blocking solution) for one hour at RT to prevent unspecific antibody binding. Subsequently, primary antibody incubation was performed overnight at 4°C in blocking solution at the following dilutions: mouse monoclonal anti-OCT4-1:200 (Santa Cruz Biotechnology Cat# sc-5279, RRID:AB_628051), rabbit polyclonal anti-RFP-1:1000 (Abcam Cat# ab124754, RRID:AB_10971665), mouse monoclonal anti-α-actinin-1:800 (Sigma-Aldrich Cat# A7811, RRID:AB_476766). Thereafter, samples were washed three times with 0.1% Tween-20 in PBS and incubated with Alexa Fluor secondary antibodies (Life Technologies) in blocking solution for one hour at RT. Afterwards, cells were washed with 0.1% Tween-20 in PBS for 3x five minutes, followed by nuclei and/or whole cell counterstaining with 0.5 µg/ml DAPI (Sigma #D9542) or Cell Mask (1:10000, Invitrogen #H32721) in PBS, respectively, for 30 minutes at RT. Samples were subsequently washed and stored at 4°C in PBS until automated image acquisition was performed in the Operetta™ high-content imaging system (PerkinElmer) and analysed using Harmony high-content imaging analysis software.\n\nLive imaging of mScarlet fluorescence in differentiating hiPSCs was performed using Operetta™ high-content image analysis every two days. All images were taken using a 20x objective. Brightfield images were taken using 100 ms exposure time. mScarlet imaging was performed using 400 ms exposure and an excitation wavelength of 520–550 nm and an emission wavelength of 560–630nm. Data analysis was performed using Columbus™ software (PerkinElmer) to identify and quantify cell regions expressing mScarlet fluorescence.\n\nReal-time qPCR reactions were performed using TaqMan® Gene Expression Assays (Applied Biosystems) following manufacturer’s instructions. Briefly, Taqman® mastermix (#4369016) including the HOXA9 probe23 was added to a MicroAmp Fast 96-well plate (#4346907). Subsequently, cDNA samples (from initial 500 ng of reverse-transcribed RNA) were added to the plate, which was thereafter sealed with a film (#4311971). Amplification was performed in ABI 7500 Real-Time PCR system (Applied Biosystems). Cycle conditions were 50°C for 2 minutes, 95°C for 10 minutes followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.). Normalisation was performed using the housekeeping gene B2M or PP1A. Relative quantification was calculated using the ΔΔCT method24 in Microsoft Excel.\n\nhiPSCs were differentiated as previously described19,20 in RPMI B27 without phenol red and dissociated on day 15 by collagenase treatment. On day 30, hiPSC-CMs were seeded at a density of 150,000 cells per well in vitronectin-N coated MatTek dishes. Intracellular Ca2+ transient measurements were made using an LSM 880C confocal microscope (Carl Zeiss) in the line-scan mode as previously described25. CMs were located using a 40x oil objective and a longitudinal line was drawn across a single CM. The R-GECO fluorophore was excited with a 561 nm laser at 0.8% power, with a detection range of 579 – 639 nm. Line-scan images were taken every 75 milliseconds, with a pixel dwell time of 4.12 µsec, for a total of 4000 cycles resulting in a five minute scan. CMs were kept at 37°C and 5% CO2 throughout data acquisition.\n\nConfocal line scan images were analysed in FiJi software, a version of ImageJ (National Institute of Health). The average fluorescence intensity of each line was calculated against time to give a confocal line-scan trace. Using the ‘multi kymograph’ function, a corresponding kymograph image was produced. In order to calculate beat rate and arrhythmic events, data was fed into pClamp software (Molecular Devices). Baselines were adjusted to account for photobleaching and Ca2+ transients were counted and analysed using the ‘event detection’ function. Using the event viewer, any Ca2+ transients that did not return to baseline and gave a ‘double peak’, or did not return to at least 75% of the previous Ca2+ transient amplitude, were considered ‘abnormal’, as described in 20.\n\nAll data presented as mean with standard deviation unless otherwise stated. Statistical analysis of multiple data sets was performed using GraphPad software (version 7.04).\n\nFor multiple comparisons between data sets a one-way ANOVA with Tukey’s multiple comparison test was chosen. For comparing multiple data sets to a single control column, one-way ANOVA with Dunnett’s multiple comparison test was chosen. Significance tests were based on p-values as follows: * p < 0.05; p < 0.01; * p < 0.001; **** p < 0.0001.\n\n\nResults\n\nOur overarching goal was to create two isogenic sets of hiPSC lines in order to study Ca2+ handling in the context of in vitro models of the disease HCM. One isogenic trio comprised lines that were wild-type (MYH7WT/WT), heterozygous (MYH7WT/MUT) and homozygous (MYH7MUT/MUT) for the c.MYH7C9123T mutation19. The other comprised a pair that were heterozygous (ACTC1WT/MUT) and corrected (ACTC1WT/WT) for the c.ACTC1G301A mutation20.\n\nThe AAVS1 locus, located within the first intron of PPP1R12C on chromosome 19 (Figure 1A), is a well characterised safe harbour locus2. Using the five lines above, we targeted a cassette containing R-GECO1.0 reporter and a puromycin resistance cassette, driven by the CAG promoter, into the AAVS1 locus (Figure 1A)22. This was achieved by using a CRISPR-Cas9 nickase approach based on two sgRNAs. Nucleofection of the HCM-associated hiPSCs with the R-GECO1.0 construct, bidirectional sgRNAs and Cas9 D10A nickase plasmids produced puromycin-resistant clones. PCR-based screening and sequencing were used to examine the regions upstream (Figure 1B) and downstream (Figure 1C) of the insertion site, and hence identify clones that were successfully targeted in one or both alleles (Figure 2B, C).\n\n(A) Schematic illustrating the chromosomal location of the AAVS1 safe harbour locus. This site was targeted using two sgRNAs in a CRISPR Cas9 nickase strategy. PAM site #1 was silently mutated (G→C) in the targeting construct to prevent it being cut by Cas9 nuclease during targeting. The inserted cassette consists of R-GECO1.0 IRES-Puromycin driven by the CAG promoter. This is flanked on each side by 1 kb of homology to the AAVS1 locus. In (B) and (C) confirmatory 5’ and 3’ targeting PCR screen is shown using genomic DNA isolated from the MYH7 C9123T RGECO1.0 isogenic trio (left) and the ACTC1 G301A RGECO duo (right) hiPSCs. Correct 5’ targeting is indicated with a 1221bp product, with sequencing confirming the fidelity of the junction between the AAVS1 left arm homology and the start of the CAG promoter. Correct 3’ targeting is indicated with an 1186bp product, with sequencing confirming the fidelity of the junction between the puromycin-SV40 pA sequence and the AAVS1 right arm homology. (D, E) Confirmatory PCR and sequencing of hiPSC clones to check 5’ and 3’ targeting of the AAVS1 locus with the HOXA9-T2A-mScarlet cassette.\n\n(A) AAVS1 targeted hiPSC clones dual-stained for OCT4 (green) and R-GECO (red) to find the highest R-GECO-expressing clone within the five cell line genotypes. High content image analysis identified that ACTC1WT/WT clone 3 had the highest percentage of pluripotent (94.53% OCT4+) and R-GECO (91.06% ±1.73%) hiPSCs. ACTC1WT/MUT clone 12 clone had the highest expression of R-GECO (49.37% ±1.33%). Mean ±SD, n = 3 wells. One-way ANOVA with Dunnett’s multiple comparison test, * p ≤ 0.0259; p < 0.0045; p < 0.0001. Scale bars = 50 µm. (B) Biallelic targeting PCR screen on isolated gDNA from targeted ACTC1WT/WT hiPSC clones showing homozygous clones failure to generate the 517bp PCR product. (C) Biallelic targeting PCR screen showing that all ACTC1WT/MUT clones tested resulted in a 517bp product and were therefore heterozygous for AAVS1 targeting. L – 1kb ladder; N – no template control; U – untargeted cell line; + - AAVS1 biallelic positive control. (D) Screening MYH7MUT/MUT clones using immunocytochemistry on differentiated hiPSC-CMs reveals a significant increase in R-GECO1.0 expression in the MYH7MUT/MUT Hom 2 clone. Mean ±SD, n = 3 technical replicates. One-way ANOVA with Tukey’s multiple comparison test, p ≤ 0.006; ** p < 0.0001. Scale bars = 50 µm.\n\nIn addition, the MYH7WT/WT hiPSC line was used to introduce a HOXA9-T2A-mScarlet cassette driven by a doxycycline-inducible pTRE promoter into the AAVS1 locus using the same CRISPR-Cas9 nickase approach (Figure 1A). Both 5’ (Figure 1D) and 3’ integration (Figure 1E) to AAVS1 was assessed using the same PCR genotyping approach on genomic DNA to identify successfully targeted clones.\n\nIn order to quantify R-GECO1.0 expression across the targeted clones, high content image analysis was used on hiPSCs that were dual-stained with anti-OCT4 for pluripotency and anti-RFP antibody, which identifies R-GECO1.0 (Figure 2A)22.\n\nTransgene expression varied widely both between, and within, cell lines. The percentage of cells expressing R-GECO1.0 in AAVS1-targeted ACTC1WT/WT hiPSC clones ranged from a maximum of 96.4% to a minimum of 32.6% (Figure 2A), and in isogenic mutant ACTC1WT/MUT hiPSC clones from 49.4% to 0%. Selected clones for the isogenic trio of MYH7WT/WT, MYH7WT/MUT and MYH7MUT/MUT hiPSCs showed comparatively high R-GECO1.0 expression exceeding 73.09% in all cases, an important requirement for a more faithful comparison between lines (Figure 2A).\n\nThis variability could not be explained by the incidence of biallelic targeting, as determined by PCR screening. Both alleles were targeted in ACTC1WT/WT clones three and six, which exhibited high R-GECO1.0 expression as hiPSCs of 91.1% and 85.4%, respectively. This was comparable with the 95.6% and 96.4% expression observed in the monoallelically targeted clones 11 and 12, respectively (Figure 2A and 2B). Variability between clones was also seen upon differentiation, with a MYH7MUT/MUT Hom 2 clone showing 93.1% R-GECO1.0 expression as hiPSC-CMs, significantly greater than the 2.1% expression observed in the MYH7MUT/MUT Hom 3 clone ( p = < 0.0001) (Figure 2D).\n\nTaken together, these results highlight that expression levels of transgenes seen in hiPSCs can vary significantly between AAVS1-targeted clones, which continued to be observed upon differentiation. Importantly, the level of variability could not be predicted and needed to be tested empirically.\n\nThree AAVS1-targeted clones of each c.MYH7C9123T genotype were identified with greater than 98.3% R-GECO1.0 expression as hiPSCs (Figure 3A and 3C)22. However, upon differentiation to cardiomyocytes, R-GECO1.0 expression significantly reduced in the biallelically targeted MYH7WT/WT clone ( p = 0.0015). The monoallelically targeted MYH7WT/MUT and MYH7MUT/MUT clones experienced considerable silencing of R-GECO1.0 expression upon differentiation, with only 13.03% of MYH7WT/MUT hiPSC-CMs and 1.33% of MYH7MUT/MUT hiPSC-CMs expressing R-GECO1.0 (** p < 0.0001) (Figure 3A–C). Nevertheless, to enhance the utility of these lines, particularly the low expressing MYH7MUT/MUT clone, puromycin enrichment of the hiPSCs over three passages was used to significantly increase the number of R-GECO1.0 expressing hiPSC-CMs from 1.3% to 18.9% (* p = 0.0003) (Figure 3D). These results show that high transgene expression from the AAVS1 locus as hiPSCs is not a guarantee of continued high expression upon differentiation, and points towards some extent of silencing upon cardiac differentiation.\n\n(A) Immunocytochemistry using an anti-RFP antibody to detect R-GECO1.0 expression shows a reduction in signal in MYH7WT/MUT 8 and MYH7MUT/MUT 15 cell lines upon differentiation from hiPSCs to hiPSC-CMs. Mean ±SD, n = 3 technical replicates. One-way ANOVA with Sidak’s multiple comparison test, p = 0.0015; p ≤ 0.0001. (B) Percentage purity data for cardiomyocytes determined by alpha-actinin staining (red), for R-GECO1.0 by RFP staining (green). Mean ±SD, n = 3 technical replicates. One-way ANOVA with Sidak’s multiple comparison test, p ≤ 0.0001. (C) Targeted hiPSC lines show variations in expression of R-GECO1.0 protein upon differentiation. hiPSC lines, identified by OCT4 staining (green, top row) show high R-GECO1.0 expression (red). Upon differentiation to cardiomyocytes, identified by α-actinin staining (red, bottom row), MYH7WT/MUT 8 and MYH7MUT/MUT 15 hiPSC-CMs show lower R-GECO1.0 expression (green). Scale bars = 50 µm. (D) R-GECO1.0 expression in MYH7MUT/MUT 15 cardiomyocytes is significantly improved with three passages of hiPSC cell culture in 0.3 µg/ml puromycin and differentiation carried out in media supplemented with puromycin. Mean ±SD, n = 3 technical replicates. Unpaired t-test, * p = 0.0003.\n\nNext, we sought to investigate the time at which silencing occurs during differentiation. To do this, we used the AAVS1-targeted doxycycline-inducible HOXA9-T2A-mScarlet line and differentiated the hiPSCs towards either cardiomyocyte or haematopoietic fate. As expected, the addition of 1 µg/ml doxycycline every 48 hours induced expression of HOXA9 and mScarlet during directed cardiac and haematopoietic differentiation. qRT-PCR analysis of HOXA9 expression showed an increase of 22738-fold higher expression compared to untargeted hiPSC control on day 0 of cardiomyocyte differentiation (Figure 4A)22. However, HOXA9 expression decreased thereafter so that by day 10, expression levels were only 175-fold greater than untargeted hiPSC control. Similarly, qRT-PCR analysis during haematopoietic differentiation revealed peak expression of HOXA9 occurring on day two, with 45666-fold greater expression than untargeted hiPSC control, decreasing thereafter to 64-fold expression on day 12 (Figure 4D). These results were mirrored at the protein level, where early peak expression of mScarlet fluorescence occurred on day 0 of cardiomyocyte differentiation (Figure 4B and 4C) and on day two of haematopoietic differentiation (Figure 4E and 4F), decreasing at later timepoints of differentiation.\n\n(A) qRT-PCR performed on hiPSCs targeted at the AAVS1 locus with a doxycycline-inducible HOXA9-T2A-mScarlet construct undergoing cardiomyocyte differentiation. Relative expression to untargeted hiPSCs. Reduced expression of the transgene is observed from day two onwards. (B) Live imaging of AAVS1-targeted hiPSCs undergoing cardiomyocyte differentiation at different timepoints. mScarlet expression peaks on day 0 and reduces throughout the differentiation, despite repeated doxycycline treatment. Scale bars = 50 µm. (C) Quantification of mScarlet expression using high content image analysis at different timepoints during cardiomyocyte differentiation. (D) qRT-PCR performed on hiPSCs targeted at the AAVS1 locus with a doxycycline-inducible HOXA9-T2A-mScarlet construct undergoing haematopoietic differentiation. Relative expression to untargeted hiPSCs. Reduced expression of the transgene is observed from day four onwards. (E) Live imaging of AAVS1-targeted hiPSCs undergoing haematopoietic differentiation at different timepoints. Scale bars = 50 µm. (F) Quantification of mScarlet expression using high content image analysis shows peak expression on day two and reduced expression thereafter. Mean ±SD, n = 2 differentiations. One-way ANOVA with Dunnett’s multiple comparison test, p = 0.0036; p ≤ 0.0001. (G, H) The mesoderm markers MIXL1 and Brachyury (T) show peak expression at day two of haematopoietic differentiation.\n\nFor the haematopoietic differentiation, expression of the key mesoderm markers MIXL1 and Brachyury peaked on day two (Figure 4G and 4H). This suggests that silencing of the AAVS1 locus can occur immediately after mesoderm patterning. As a whole, these results show a progressive silencing of transgene expression as mesoderm differentiation progresses.\n\nDespite some obstacles due to unanticipated AAVS1 silencing, isogenic R-GECO1.0 expressing clones in genetic backgrounds associated with HCM were successfully generated and used to image Ca2+ transients using confocal laser line scan microscopy. As this technique involves assaying single cells, even poorly expressing clones were useful. By monitoring the fluctuation of R-GECO1.0 fluorescence over time, Ca2+ transient traces could be generated for each line (Figure 5A–C and 5E–F)22. For both the c.MYH7C9123T and c.ACTC1G301A mutations, increasing mutation load resulted in an increased incidence of abnormal Ca2+ transient events. MYH7WT/WT hiPSC-CMs only presented 1.1% aberrant Ca2+ transient events, increasing to 4.33% in MYH7WT/MUT hiPSC-CMs, and further increasing to 11.2% in MYH7MUT/MUT hiPSC-CMs (p < 0.0001) (Figure 5D). This represented a ten-fold increase in the occurrence of aberrant Ca2+ transients in the homozygous mutant compared to isogenic wild-type control. Likewise, 15.65% of Ca2+ transients in ACTC1WT/MUT hiPSC-CMs were calculated as being aberrant, compared to 6.7% (±0.6%) in ACTC1WT/WT isogenic control hiPSC-CMs (p = 0.0118) (Figure 5G). This demonstrated the utility of the AAVS1-targeted R-GECO1.0 cell lines for in vitro disease modelling and phenotyping as a credible alternative to the use of animal models.\n\n(A–C) Representative 25-second confocal laser line scan traces and kymographs for isogenic trio of c.MYH7C9123T R-GECO1.0 expressing hiPSC-CMs at day 30. Abnormal Ca2+ transient events (red arrows) increase in frequency with mutation load. (D) Ca2+ transient event detection and quantification showing percentage of Ca2+ transients deemed abnormal. Data presented as mean ±SD, n = 6 scans across three differentiations. Kruskal-Wallis test with Dunn’s multiple comparison test; p = 0.0018, **** p < 0.0001. (E–F) Representative 25-second confocal laser line scan traces and kymographs for isogenic pair of c.ACTC1G301A R-GECO1.0 expressing hiPSC-CMs at day 30. Abnormal Ca2+ transient events (red arrows) increase in frequency with mutation load. (G) Box-plot showing % of total Ca2+ transient events detected deemed abnormal by event detection software. Data presented as mean ±SD, n = 5 scans from three differentiations. Unpaired t-test; * p = 0.0118. (H–L) Representative line scans and event detection quantification showing a reduction in abnormal Ca2+ transient events upon treatment with 10 µM ranolazine and 10 µM dantrolene compared to 0.1% DMSO vehicle control. Data presented as mean ±SD, n > 4 cells across minimum of two differentiations. Kruskal-Wallis test with Dunn’s multiple comparison test; ** p = 0.0068.\n\nWe then attempted to rescue the aberrant Ca2+ transient phenotype in our HCM models with the use of a combination treatment of ranolazine, a late sodium channel blocker, and dantrolene, a ryanodine receptor antagonist. In combination at 10 µM with 24 hours incubation, these two drugs significantly reduced the frequency of aberrant Ca2+ transient events in MYH7 mutant hiPSC-CMs. In MYH7WT/MUT hiPSC-CMs aberrant Ca2+ transient event frequency was reduced from 6.32% (±3.4%) in vehicle control to 2.18% (±1.9%) with drug treatment (p = 0.0068) (Figure 5H–I).\n\nThese results show that abnormalities in Ca2+ transients caused by the sarcomeric mutations c.MYH7C9123T or c.ACTC1G301A can be identified with AAVS1-targeted R-GECO1.0 expression, and this phenotype can be subsequently rescued with targeted pharmacological intervention aimed at reducing intracellular Na+ and Ca2+.\n\n\nDiscussion\n\nPrecise integration of exogenous DNA into the genome is often performed by targeting a genomic ‘safe harbour’ locus that can tolerate gene insertion with few deleterious effects and limited transgene silencing. The AAVS1 locus is a popular choice for targeted knock in of exogenous DNA15,16. This region of the genome is claimed to facilitate robust and persistent transgene expression11, aided by flanking insulator regions10. Here, we show variable success targeting the AAVS1 locus with the genetically encoded calcium indicator R-GECO1.0 or a doxycycline-inducible HOXA9-T2A-mScarlet cassette using a CRISPR Cas9 nickase approach.\n\nVariability in R-GECO1.0 expression between AAVS1-targeted clones as hiPSCs was observed, highlighting the importance of thorough screening of clones. Unsurprisingly, clones that had undergone biallelic targeting retained high R-GECO1.0 expression as hiPSCs, yet monoallelically targeted clones ranged from high expression to significantly reduced R-GECO1.0 expression. These incidences of low expression as hiPSCs may be due to clone-specific silencing, or some clones favouring expression from the untargeted allele. It has been shown that some genes within cells favour monoallelic expression26. Indeed, our own studies using an antibody for the c.ACTC1G301A mutation have shown that cells heterozygous for the mutation only express mutant protein in ~50% of the population20. These results highlight the heterogeneity that can exist between clones once they have been generated.\n\nEven with the identification of AAVS1-targeted clones that exhibited robust R-GECO1.0 expression, there were instances of silencing upon differentiation to hiPSC-CMs. Transgene silencing at the AAVS1 locus has previously been shown upon differentiation towards hepatocyte-like cells, with de novo methylation of the locus found to be responsible12. However, the aforementioned report claims that this silencing effect is restricted to endoderm differentiations. With the use of the AAVS1-targeted HOXA9-T2A-mScarlet cell line, we show that in two mesoderm-specific differentiations towards cardiomyocytes and haematopoietic cells, silencing of expression occurs immediately after mesoderm specification and peak expression of MIXL1 and Brachyury. We therefore postulate that AAVS1-mediated silencing, likely as a result of de novo methylation, can occur upon differentiation to various germ layers, and therefore AAVS1 cannot be considered a true safe harbour locus.\n\nThe choice of promoter may also play a role in AAVS1-mediated silencing, as a previous report has shown AAVS1 silencing of eGFP expression when using the EF1a promoter that was overcome with the use of the stronger CAG promoter27. This influenced our decision to opt for the CAG promoter over the EF1a promoter in our constructs. Indeed, the CAG promoter appears to exhibit some insulation from methylation of transgenes at the AAVS1 locus12.\n\nWhen attempting to express two separate transgenes from the same cassette at the AAVS1 locus, the choice of peptide cleavage sequence is also important. We observed inefficient peptide cleavage when using the P2A sequence, as determined by Western Blot (see Extended data)22. This informed our choice of using the T2A or IRES sequences for translation of multicistronic cassettes in subsequent targeting constructs. It has been claimed that the P2A cleavage sequence is the most efficient self-cleaving peptide, followed by T2A and E2A, when used to cleave a bicistronic vector in three different human cell lines, including HeLa28. We cannot reconcile this with our data, as co-expression of multicistronic cassette elements from the AAVS1 locus in hiPSCs has been achieved using the IRES and T2A sequence, but not the P2A sequence.\n\nDepending on the application of the targeted cell lines, some AAVS1-mediated silencing can be tolerated. For the R-GECO1.0 expressing lines, maintaining puromycin selection and using a single cell confocal laser line scan assay to study Ca2+ transients meant that abnormalities in Ca2+ handling could be identified in cell lines harbouring the c.MYH7C9123T or c.ACTC1G301A mutations. This represents an in vitro model of HCM that can offer an alternative to the use of animal models. Furthermore, this phenotype could be rescued with combination treatment with dantrolene and ranolazine19,20. However, the aim with the doxycycline-inducible HOXA9-T2A-mScarlet cell line was to modulate HOXA9 expression at different timepoints throughout haematopoietic differentiation. Clearly, the clone described herein is not suitable for this task.\n\nIn conclusion, one must carefully select AAVS1-targeted clones depending on their application due to the risk of transgene silencing upon differentiation. Other potential safe harbour loci, such as CLYBL, have been claimed to deliver five- to ten-fold higher fluorescent transgene expression than AAVS16. However, as silencing cannot be predicted, multiple clones must be thoroughly checked for expression level and chosen according to their application. Our results dispute the claims of robust and persistent transgene expression from AAVS111, and complements reports that show silencing at AAVS1 upon differentiation to endoderm lineage12, by showing similar silencing upon mesoderm differentiation.\n\n\nData availability\n\nFigShare: Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour. https://doi.org/10.6084/m9.figshare.c.4573316.v222\n\nThis project contains the following underlying data:\n\n- Figure 1 – data (raw PCR integration gel images in TIF format; and sequencing chromatograms underlying Figure 1 as AB1 files)\n\n- Figure 2 – data (raw immunocytochemistry images in PNG format; PCR gel images in TIF and PNG format; and XLS files containing immunocytochemistry expression quantification data underlying Figure 2)\n\n- Figure 3 – data (raw immunocytochemistry images in PNG format; and XLS files containing expression quantification data underlying Figure 3)\n\n- Figure 4 – data (raw live imaging images in PNG format; XLS files containing qPCR data underlying Figure 4)\n\n- Figure 4 data – Raw qPCR data (XLS files containing raw qPCR data)\n\n- Figure 5 – data (kymographs in PNG format; XLS files containing raw confocal laser line scan data underlying Figure 5)\n\nFigShare: Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour. https://doi.org/10.6084/m9.figshare.c.4573316.v222\n\nThis project contains the following extended data:\n\n- Figure 6. Western Blot to show incomplete cleavage of multicistronic cassette when using the P2A peptide cleavage sequence (TIF image file)\n\n- Protocol for CRISPR Cas9 knock-in at the AAVS1 locus and subsequent screening (a step-by-step procedure for performing CRISPR Cas9 knock-in at the AAVS1 locus of hiPSCs, followed by subsequent screening techniques in DOCX format)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Acknowledgements\n\nThe original Myc-P2A-R-GECO1.0 construct plasmid was received as a kind gift from Dr Matt Daniels at The University of Manchester17. hCas9_D10A was a gift from Professor George Church (Addgene plasmid #41816).\n\n\nReferences\n\nPapapetrou EP, Schambach A: Gene Insertion Into Genomic Safe Harbors for Human Gene Therapy. Mol Ther. 2016; 24(4): 678–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSadelain M, Papapetrou EP, Bushman FD: Safe harbours for the integration of new DNA in the human genome. Nat Rev Cancer. 2011; 12(1): 51–8. PubMed Abstract \| Publisher Full Text\n\nLombardo A, Genovese P, Beausejour CM, et al.: Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat Biotechnol. 2007; 25(11): 1298–306. PubMed Abstract \| Publisher Full Text\n\nvan Rensburg R, Beyer I, Yao XY, et al.: Chromatin structure of two genomic sites for targeted transgene integration in induced pluripotent stem cells and hematopoietic stem cells. Gene Ther. 2013; 20(2): 201–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nIrion S, Luche H, Gadue P, et al.: Identification and targeting of the ROSA26 locus in human embryonic stem cells. Nat Biotechnol. 2007; 25(12): 1477–82. PubMed Abstract \| Publisher Full Text\n\nCerbini T, Funahashi R, Luo Y, et al.: Transcription activator-like effector nuclease (TALEN)-mediated CLYBL targeting enables enhanced transgene expression and one-step generation of dual reporter human induced pluripotent stem cell (iPSC) and neural stem cell (NSC) lines. PLoS One. 2015; 10(1): e0116032. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSamulski RJ, Zhu X, Xiao X, et al.: Targeted integration of adeno-associated virus (AAV) into human chromosome 19. EMBO J. 1991; 10(12): 3941–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeister T, Heid I, Ackermann M, et al.: Herpes simplex virus type 1/adeno-associated virus hybrid vectors mediate site-specific integration at the adeno-associated virus preintegration site, AAVS1, on human chromosome 19. J Virol. 2002; 76(14): 7163–73. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHockemeyer D, Soldner F, Beard C, et al.: Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol. 2009; 27(9): 851–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOgata T, Kozuka T, Kanda T: Identification of an insulator in AAVS1, a preferred region for integration of adeno-associated virus DNA. J Virol. 2003; 77(16): 9000–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith JR, Maguire S, Davis LA, et al.: Robust, persistent transgene expression in human embryonic stem cells is achieved with AAVS1-targeted integration. Stem Cells. 2008; 26(2): 496–504. PubMed Abstract \| Publisher Full Text\n\nOrdovás L, Boon R, Pistoni M, et al.: Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition. Stem Cell Reports. 2015; 5(5): 918–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang J, DeClercq JJ, Hayward SB, et al.: Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery. Nucleic Acids Res. 2016; 44(3): e30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLombardo A, Cesana D, Genovese P, et al.: Site-specific integration and tailoring of cassette design for sustainable gene transfer. Nat Methods. 2011; 8(10): 861–9. PubMed Abstract \| Publisher Full Text\n\nYada RC, Ostrominski JW, Tunc I, et al.: CRISPR/Cas9-Based Safe-Harbor Gene Editing in Rhesus iPSCs. Curr Protoc Stem Cell Biol. 2017; 43: 5A.11.1–5A.11.14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCastaño J, Bueno C, Jiménez-Delgado S, et al.: Generation and characterization of a human iPSC cell line expressing inducible Cas9 in the \"safe harbor\" AAVS1 locus. Stem Cell Res. 2017; 21: 137–140. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChang YF, Broyles CN, Brook FA, et al.: Non-invasive phenotyping and drug testing in single cardiomyocytes or beta-cells by calcium imaging and optogenetics. PLoS One. 2017; 12(4): e0174181. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSparrow AJ, Sievert K, Patel S, et al.: Measurement of Myofilament-Localized Calcium Dynamics in Adult Cardiomyocytes and the Effect of Hypertrophic Cardiomyopathy Mutations. Circ Res. 2019; 124(8): 1228–1239. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMosqueira D, Mannhardt I, Bhagwan JR, et al.: CRISPR/Cas9 editing in human pluripotent stem cell-cardiomyocytes highlights arrhythmias, hypocontractility, and energy depletion as potential therapeutic targets for hypertrophic cardiomyopathy. Eur Heart J. 2018; 39(43): 3879–3892. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmith JGW, Owen T, Bhagwan JR, et al.: Isogenic Pairs of hiPSC-CMs with Hypertrophic Cardiomyopathy/LVNC-Associated ACTC1 E99K Mutation Unveil Differential Functional Deficits. Stem Cell Reports. 2018; 11(5): 1226–1243. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBreckwoldt K, Letuffe-Brenière D, Mannhardt I, et al.: Differentiation of cardiomyocytes and generation of human engineered heart tissue. Nat Protoc. 2017; 12(6): 1177–1197. PubMed Abstract \| Publisher Full Text\n\nBhagwan J, Collins E, Mosqueira D, et al.: Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour. figshare. Collection. 2019. http://www.doi.org/10.6084/m9.figshare.c.4573316.v2\n\nDickson GJ, Lappin TR, Thompson A: Complete array of HOX gene expression by RQ-PCR. Methods Mol Biol. 2009; 538: 369–93. PubMed Abstract \| Publisher Full Text\n\nSchmittgen TD, Livak KJ: Analyzing real-time PCR data by the comparative CT method. Nat Protoc. 2008; 3(6): 1101–1108. PubMed Abstract \| Publisher Full Text\n\nYazawa, M, Hsueh B, Jia X, et al.: Using induced pluripotent stem cells to investigate cardiac phenotypes in Timothy syndrome. Nature. 2011; 471(7337): 230–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEckersley-Maslin MA, Spector DL: Random monoallelic expression: regulating gene expression one allele at a time. Trends Genet. 2014; 30(6): 237–44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLuo Y, Liu C, Cerbini T, et al.: Stable enhanced green fluorescent protein expression after differentiation and transplantation of reporter human induced pluripotent stem cells generated by AAVS1 transcription activator-like effector nucleases. Stem Cells Transl Med. 2014; 3(7): 821–35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim JH, Lee SR, Li LH, et al.: High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One. 2011; 6(4): e18556. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "56469", "date": "29 Nov 2019", "name": "Carolyn Carr", "expertise": [ "Reviewer Expertise Cardiac physiology and cardiac stem cells for regeneration", "disease phenotyping and drug testing." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 are two arms to this manuscript. The authors aimed to determine the reliability of introducing a transgene containing a calcium indicator (R-GECO1.0) or a fluorescent reporter (HOXA9-T2A-mScarlet) into the AAVS1 locus using CRISPR-Cas9 and then subsequently investigate the effect of mutations in MYH7 or ACTC1 on calcium handling. Initial experiments showed that although the AAVS1 could be targeted successfully in 20/24 clones, the expression of the transgene was highly variable. Furthermore, on differentiation of the more successfully transfected clones to cardiomyocytes, the transgene was silenced with almost complete removal of expression of the fluorescent reporter. A similar degree of silencing was observed during haematopoetic differentiation. Using the mScarlet reporter the authors investigated the timecourse of downregulation and found that with both cardiomyocyte and haematopoetic differentiation, increased transgene expression was observed at day 2 but then decreased substantially over time, at both the mRNA and protein level. Nevertheless, using puromycin selection, it was possible to isolate single cardiomyocytes expressing the R-GECO reporter and these were used to show that mutations in both MYH7 and ACTC1 resulted in abnormal calcium transient events which could be corrected in part using ranolazine and dantrolene.\n\nThis is a valuable piece of work which demonstrates shortfalls in what was expected to be a fairly reliable transfection protocol and that the AAVS1 locus is not as foolproof a site for transfection as may have been thought. The addition of the calcium transient measurements is interesting in that it shows that something can be rescued from such a large body of work, but it does come as rather an afterthought in the manuscript.\n\nI have a few minor comments to aid clarity:\nThe introduction needs to be expanded somewhat. The paragraph on modulating HOXA9 is very brief and the rationale needs to be explained. In the results section this appears to be merely a way of monitoring transgene expression, but the short paragraph in the introduction implies some sort of mechanistic approach.\n\nIt is not entirely clear to me what the difference is between the data shown in Fig 2D and 3B apart from different clones.\n\nIn the section on HOXA9-T2A targeting it says in results that doxycycline was administered every 48 hours. This is not mentioned in the methods or the extended information. The authors state that transgene induction decreased after day 2. Was this reduction despite further addition of doxycycline?\n\nIn figure 5, the timing on the x axis is given in parts H and I but not in other plots. In some others there is a bar but this is not explained. Is the scale the same throughout? If so, some comment should be made as to why the wild type MYH7 cells have a slower beat rate than the ACTC1 wild type cells. The same formatting should be used on all the graphs.\n\nIs the work 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": "5537", "date": "10 Jul 2020", "name": "Jamie Bhagwan", "role": "Author Response", "response": "Dear Reviewer, Thank you for your comments on our manuscript entitled Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour. We have endeavoured to respond to your comments and suggestions as outlined below in bold font. There are two arms to this manuscript. The authors aimed to determine the reliability of introducing a transgene containing a calcium indicator (R-GECO1.0) or a fluorescent reporter (HOXA9-T2A-mScarlet) into the AAVS1 locus using CRISPR-Cas9 and then subsequently investigate the effect of mutations in MYH7 or ACTC1 on calcium handling. Initial experiments showed that although the AAVS1 could be targeted successfully in 20/24 clones, the expression of the transgene was highly variable. Furthermore, on differentiation of the more successfully transfected clones to cardiomyocytes, the transgene was silenced with almost complete removal of expression of the fluorescent reporter. A similar degree of silencing was observed during haematopoetic differentiation. Using the mScarlet reporter the authors investigated the timecourse of downregulation and found that with both cardiomyocyte and haematopoetic differentiation, increased transgene expression was observed at day 2 but then decreased substantially over time, at both the mRNA and protein level. Nevertheless, using puromycin selection, it was possible to isolate single cardiomyocytes expressing the R-GECO reporter and these were used to show that mutations in both MYH7 and ACTC1 resulted in abnormal calcium transient events which could be corrected in part using ranolazine and dantrolene. This is a valuable piece of work which demonstrates shortfalls in what was expected to be a fairly reliable transfection protocol and that the AAVS1 locus is not as foolproof a site for transfection as may have been thought. The addition of the calcium transient measurements is interesting in that it shows that something can be rescued from such a large body of work, but it does come as rather an afterthought in the manuscript. I have a few minor comments to aid clarity: The introduction needs to be expanded somewhat. The paragraph on modulating HOXA9 is very brief and the rationale needs to be explained. In the results section this appears to be merely a way of monitoring transgene expression, but the short paragraph in the introduction implies some sort of mechanistic approach. For the purposes of this paper the HOXA9 model is simply used a tool to monitor transgene expression. However, its overall purpose in the context of haematopoiesis is now described in the introduction for completeness. The penultimate paragraph of the Introduction now reads: “In addition, CRISPR Cas9 targeting of the AAVS1 locus was used to target a doxycycline-inducible HOXA9-T2A-mScarlet cassette into hiPSCs. HOXA9 is a transcription factor regulated spatio-temporally during haematopoietic or cardiac development (Behrens et al., 2013) and the aim was to examine if controlled supplemental expression of HOXA9 resulted in more efficient production of mature cells.” It is not entirely clear to me what the difference is between the data shown in Fig 2D and 3B apart from different clones. Yes, we concede that these experiments are similar. However, Fig 2D is intended to show variability within an isogenic set of clones differentiated to cardiomyocytes, whereas Fig 3B is intended as a comparison between isogenic sets of clones and complements the data in Fig 3A. In the section on HOXA9-T2A targeting it says in results that doxycycline was administered every 48 hours. This is not mentioned in the methods or the extended information. The authors state that transgene induction decreased after day 2. Was this reduction despite further addition of doxycycline? Yes, this reduction did occur despite further addition of doxycycline every 2 days. In addition to text in the results and the figure legend for Figure 3, the methods have now been clarified with a sentence in the ‘Live imaging of mScarlet’ section which now reads: “HOXA9 and mScarlet expression was induced with the addition of 1 µg/ml doxycline every 48 hours.” In figure 5, the timing on the x axis is given in parts H and I but not in other plots. In some others there is a bar but this is not explained. Is the scale the same throughout? If so, some comment should be made as to why the wild type MYH7 cells have a slower beat rate than the ACTC1 wild type cells. The same formatting should be used on all the graphs. The authors apologise for the error. The x axis scale bar relates to time (5 seconds) and the y-axis scale bar relates to the length of the laser line drawn across the cell to perform the confocal line scan. All panels now contain both scale bars and the legend for Figure 5 has been corrected to include this information. The methods have also been clarified to state that these line scans are performed on spontaneously beating cardiomyocytes, hence their slightly varied beat rate. The end of the first paragraph of the ‘confocal analysis’ methods sections now reads: “Line-scan images were taken every 75 milliseconds, with a pixel dwell time of 4.12 µsec, for a total of 4000 cycles resulting in a five minute scan. CMs were kept at 37°C and 5% CO 2 and allowed to spontaneously beat throughout data acquisition.” Differences in beat rate and action potential duration between healthy hiPSC-CMs are common, as previously reviewed (Sala et al., 2017). This further advocates the need for isogenic lines in order to ensure that the impact of the mutation studied is accurately investigated. The fourth sentence of the section entitled “AAVS1-targeted R-GECO1.0 expressing clones as a tool for in vitro disease modelling and drug screening” now reads: “Despite some expected variability in spontaneous beat rate between wild-type hiPSC-CMs (Figure 5A and 5E) (Sala et al., 2017), for both the c. MYH7 C9123T and c. ACTC1 G301A mutations, increasing mutation load resulted in an increased incidence of abnormal Ca 2+ transient events.” References Behrens AN, Iacovino M, Lohr JL, et al.: Nkx2-5 mediates differential cardiac differentiation through interaction with Hoxa10. Stem Cells Dev. 2013;22(15):2211-2220. 23477547 10.1089/scd.2012.0611 Sala L, Bellin M, Mummery CL.: Integrating cardiomyocytes from human pluripotent stem cells in safety pharmaology: Has the time come? Br J Pharmacol. 2017;174(21):3749-3765. 27641943 10.1111/bph.13577" } ] }, { "id": "61069", "date": "23 Mar 2020", "name": "Catherine M. Verfaillie", "expertise": [ "Reviewer Expertise iPSC", "differentiation", "genome editing" ], "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 paper, the authors aim to investigate the effect of hypertrophic cardiomyopathy associated mutations MYH7C9123T and ACTC1G301A on Ca2+ transients using hiPSCs- derived cardiomyocytes. To this end, the authors established hiPSCs reporters by introducing a CAG promotor-controlled calcium indicator (R-GECO1.0) into the AAVS1 locus through CRISPR/Cas9 nickase-mediated genome editing. Among 24 clones, 20 were found to successfully express the transgene with variation from about 0 to 99.5%. Upon differentiation to cardiomyocytes, even clones with high expression levels demonstrated significant silencing of the transgene to 13.03% or 1.33%. By creating also an AAVS1 targeted doxycycline inducible HOXA9-T2A-mScarlet iPSCs reporter, the authors investigated the silencing over the time course during mesoderm lineage commitment. It was shown that both mRNA and protein levels of the transgenes were relatively high on day 2 and abruptly decreased from day 4 during both cardiomyocyte and hematopoietic differentiation. Despite the silencing of R-GECO1.0 in the AAVS1 locus, Ca2+ live image by confocal laser line scan microscopy at the single cell level detected abnormal Ca2+ transients in cardiomyocytes derived from hiPSCs reporters harboring MYH7C or ACTC1 mutations compared to wildtype or isogenic control. In addition, this abnormality could be rescued by pharmacological inhibiting intercellular level of Na+ and Ca2+. This study shows that iPSC reporter line is of importance for disease modelling. This study presented an important issue in AAVS1-targeted transgene expression in iPSCs and mesoderm lineage although the mechanisms for the silencing are not clear. The variable expression and silencing in mesoderm differentiation shown here are in line with previous reports that the AAVS1 is not a true safe harbor for cells differentiated to hematopoietic cells (e.g. PMID: 31773990) and endoderm (e.g. PMID: 26455413); and, hence, the findings are not fully novel.\nSpecific comments:\nThe authors did not mention why for they compared wild-type (MYH7WT/WT), heterozygous (MYH7WT/MUT) and homozygous (MYH7MUT/MUT) for the c.MYH7C9123T mutation, but no homozygous mutant line for ACTC1G301A.\n\nIn Fig 2A, the expression of R-GECO in ACTC1WT/MUT clones (1/5 with about 50% cells) was lower than in ACTC1WT/WT clones. Is this chance or due to the mutation?\n\nIn Fig 2A, it would be better to show R-GECO in red and OCT4 in green to retain consistency with other images.\n\nIn Fig 3C, immunostaining in the upper images showed R-GECO in red while in the lower ones R-GECO is stained in green, which is difficult to follow.\n\nIt is interesting to see that puromycin enrichment of the iPSCs over 3 passages increased R-GECO1.0 expression in MYH7MUT/MUT 15, did the author also tried puromycin enrichment in MYH7WT/MUT?\n\nIn the introduction, the authors mention that a doxycycline-inducible HOXA9-T2A-mScarlet cassette targeted in the AAVS1 locus of hiPSCs is used for modulating HOXA9 during hematopoietic differentiation. However, this iPSC reporter is also used for cardiomyocyte differentiation (Fig 4A). It is unclear whether there is specific reason why this reporter line is used instead of AAVS1-CAG R-GECO iPSCs.\n\nIt is better to present RTqPCR result using a ∆Ct method for Fig. 4 as fold changes are confusing especially in the context of transgene expression (the biological relevance of fold change is unclear unless one knows the base line transcript levels before gene activation).\n\nIn Fig 6 - Western Blot (Extended data), to test the peptide cleavage of P2A and T2A, the authors state that P2A is less efficient than T2A, as NpHR expression (following a P2A) was barely visible. The authors should provide positive control to exclude that the antibody for NpHR did not work.\n\nSouthern blots should be performed to make sure that clones tested in this study were targeted in the corrected locus, and silencing was not due to random integration.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "5538", "date": "10 Jul 2020", "name": "Jamie Bhagwan", "role": "Author Response", "response": "Dear Reviewer, Thank you for your comments on our manuscript entitled Variable expression and silencing of CRISPR-Cas9 targeted transgenes identifies the AAVS1 locus as not an entirely safe harbour.We have endeavoured to respond to your comments and suggestions as outlined below in bold font. In this paper, the authors aim to investigate the effect of hypertrophic cardiomyopathy associated mutations MYH7C9123T and ACTC1G301A on Ca2+ transients using hiPSCs- derived cardiomyocytes.To this end, the authors established hiPSCs reporters by introducing a CAG promotor-controlled calcium indicator (R-GECO1.0) into the AAVS1 locus through CRISPR/Cas9 nickase-mediated genome editing. Among 24 clones, 20 were found to successfully express the transgene with variation from about 0 to 99.5%. Upon differentiation to cardiomyocytes, even clones with high expression levels demonstrated significant silencing of the transgene to 13.03% or 1.33%. By creating also an AAVS1 targeted doxycycline inducible HOXA9-T2A-mScarlet iPSCs reporter, the authors investigated the silencing over the time course during mesoderm lineage commitment. It was shown that both mRNA and protein levels of the transgenes were relatively high on day 2 and abruptly decreased from day 4 during both cardiomyocyte and hematopoietic differentiation.Despite the silencing of R-GECO1.0 in the AAVS1 locus, Ca2+ live image by confocal laser line scan microscopy at the single cell level detected abnormal Ca2+ transients in cardiomyocytes derived from hiPSCs reporters harboring MYH7C or ACTC1 mutations compared to wildtype or isogenic control. In addition, this abnormality could be rescued by pharmacological inhibiting intercellular level of Na+ and Ca2+.This study shows that iPSC reporter line is of importance for disease modelling. This study presented an important issue in AAVS1-targeted transgene expression in iPSCs and mesoderm lineage although the mechanisms for the silencing are not clear. The variable expression and silencing in mesoderm differentiation shown here are in line with previous reports that the AAVS1 is not a true safe harbor for cells differentiated to hematopoietic cells (e.g. PMID: 31773990) and endoderm (e.g. PMID: 26455413); and, hence, the findings are not fully novel.We note the reviewer’s comments regarding novelty of the findings. However, the Klatt et al. paper (PMID: 31773990) was published after the submission of this manuscript. The Klatt et al. paper was first published online on 27 November 2019, whilst this manuscript was first published on 12 November 2019, so the variable expression and silencing upon hematopoietic differentiation was undocumented prior to the submission of this manuscript. Nonetheless, the Klatt et al paper adds weight to the notion of AAVS1 silencing, and elegantly demonstrates the contextual methylation of AAVS1-targeted transgenes depending on the promoter inserted into the site. We also note the Luo et al 2014 paper (PMID: 24833591) which showed silencing of eGFP expression at the AAVS1 site when using the EF1α promoter that was overcome by replacing it with the CAG promoter. Indeed, this paper informed our choice of promoter for the RGECO targeting construct. The penultimate sentence of the third paragraph in the Introduction now reads:“However, some reports of DNA methylation dampening transgene expression in both hPSC-derived hepatocytes (Ordovás et al., 2015) and iPSC-derived myeloid progenitors (Klatt et al., 2020) raise questions on whether a ‘perfect’ safe harbour locus exists” The Discussion now reads: “Transgene silencing at the AAVS1 locus has previously been shown upon differentiation towards hepatocyte-like cells, with de novo methylation of the locus found to be responsible (Ordovás et al., 2015). However, the aforementioned report claims that this silencing effect is restricted to endoderm differentiations. With the use of the AAVS1-targeted HOXA9-T2A-mScarlet cell line, we show that in two mesoderm-specific differentiations towards cardiomyocytes and haematopoietic cells, silencing of expression occurs immediately after mesoderm specification and peak expression of MIXL1 and Brachyury. This is in agreement with a recent report which demonstrated differential transgene methylation in AAVS1-targeted iPSC-derived myeloid cells (Klatt et al., 2020).” And “Indeed, the CAG promoter appears to exhibit some insulation from methylation of transgenes at the AAVS1 locus (Ordovás et al., 2015). In addition, a recent report elegantly demonstrated that contextual silencing at the AAVS1 locus of iPSC-derived myeloid precursors can occur with varying efficiency depending on the chosen promoter (Klatt et al., 2020).” The authors recognise and acknowledge that this phenomenon has been shown in endoderm and this is referred to in the manuscript. The last paragraph of the Introduction contains the following sentence: “This suggests that silencing at the AAVS1 locus is not limited to the endoderm lineage as previously described (Ordovás et al., 2015). The Discussion states that: “Our results dispute the claims of robust and persistent transgene expression from AAVS1, and complements reports that show silencing at AAVS1 upon differentiation to endoderm lineage (Ordovás et al., 2015)” Specific comments: The authors did not mention why for they compared wild-type (MYH7WT/WT), heterozygous (MYH7WT/MUT) and homozygous (MYH7MUT/MUT) for the c.MYH7C9123T mutation, but no homozygous mutant line for ACTC1G301A.The authors apologise for not making this clearer in the text. The discrepancy between the range of genetically-engineered lines exhibiting the MYH7- or ACTC1- mutations is due to the source of the cells and their subsequent CRISPR/Cas9 targeting strategy. The MYH7-mutant hiPSC lines were generated by targeting the WT allele of unrelated healthy cell lines or origin (Mosqueira et al., 2018), enabling the generation of heterozygous and homozygous clones. In contrast, the isogenic set of ACTC1 lines was generated by genomic correction of the mutant allele of the starting hiPSC line derived from a heterozygous patient (using a donor vector containing the WT allele only) (Smith et al., 2018, Kondrashov et al., 2018). As such, homozygous mutant clones could not be generated by employing this strategy. Notwithstanding, the vast majority of HCM-causing mutations in the sarcomeric genes are heterozygous (Lopes et al, 2013), as homozygous mutations tend to be lethal. In particular, the mutations under study (p.R453C-βMHC and p.E99K-ACTC1) have never reported to be homozygous in patients, to the best of our knowledge. Therefore, the cellular models generated already encompass the patient-relevant genotypic status and as such provide an accurate characterization of HCM in vitro. Homozygous mutant lines were included to provide extra readout sensitivity for the phenotypic assays developed (as seen by more severe phenotypes in Figure 5).The first paragraph of the Results section now reads: “Our overarching goal was to create two isogenic sets of hiPSC lines in order to study Ca2+ handling in the context of in vitro models of the disease HCM. One isogenic trio comprised lines that were originally wild-type (MYH7 WT/WT) and then CRISPR Cas9 edited to generate heterozygous (MYH7 WT/MUT) and homozygous (MYH7 MUT/MUT) mutants for the c. MYH7 C9123T mutation (Mosqueira et al., 2018) . The other comprised a pair that were originally patient-derived heterozygous (ACTC1 WT/MUT) for the c. ACTC1 G301A mutation and CRISPR Cas9 corrected (ACTC1 WT/WT) (Smith et al., 2018, Kondrashov et al., 2018). In Fig 2A, the expression of R-GECO in ACTC1WT/MUT clones (1/5 with about 50% cells) was lower than in ACTC1WT/WT clones. Is this chance or due to the mutation?We believe that this is unlikely to be due to the ACTC1 mutation but simply highlights the variability between different cell lines. Each cell line, and subsequently, each clone seemingly has varying levels of transgene silencing and extensive screening is therefore required, as we have performed. In Fig 2A, it would be better to show R-GECO in red and OCT4 in green to retain consistency with other images.Agreed. The images in Figure 2A for MYH7MUT/MUT clone 1 and MYH7MUT/MUT clone 4 have now been pseudocoloured to match the other images in the panel. In Fig 3C, immunostaining in the upper images showed R-GECO in red while in the lower ones R-GECO is stained in green, which is difficult to follow.Agreed. To retain consistency, the upper images in Figure 3C have been pseudocoloured so that R-GECO is always represented as red. Figure 2D and all accompanying graphs have also been changed to aid consistency. It is interesting to see that puromycin enrichment of the iPSCs over 3 passages increased R-GECO1.0 expression in MYH7MUT/MUT 15, did the author also tried puromycin enrichment in MYH7WT/MUT?Our aim was to have a high enough percentage of cardiomyocytes expressing R-GECO so that confocal line scan analysis was technically feasible. The MYH7MUT/MUT 15 clone was originally enriched due to the extremely low level of cardiomyocyte R-GECO expression making it difficult to perform single cell analysis. In contrast, the 13.03% R-GECO expression in the MYH7WT/MUT clone was sufficient to find R-GECO expressing cardiomyocytes in a single field of view. Therefore, antibiotic selection of the MYH7WT/MUT line was not necessary. The purpose of Figure 3D is to provide a technical solution to lower levels or R-GECO expression, when required. In the introduction, the authors mention that a doxycycline-inducible HOXA9-T2A-mScarlet cassette targeted in the AAVS1 locus of hiPSCs is used for modulating HOXA9 during hematopoietic differentiation. However, this iPSC reporter is also used for cardiomyocyte differentiation (Fig 4A). It is unclear whether there is specific reason why this reporter line is used instead of AAVS1-CAG R-GECO iPSCs.We acknowledge the Reviewer’s point. As cardiomyocyte and haematopoietic differentiation share developmental stages (e.g., mesoderm induction), and given that HOXA9 was also shown to be expressed in early cardiac progenitor cells of the cardiac crescent (Behrens et al., 2013), we opted to perform both cardiomyocyte and haematopoietic differentiations using the HOXA9-T2A-mScarlet line. The introduction has been amended to clarify the overall purpose of the HOXA9-T2A-mScarlet line: “In addition, CRISPR Cas9 targeting of the AAVS1 locus was used to target a doxycycline-inducible HOXA9-T2A-mScarlet cassette into hiPSCs. HOXA9 is a transcription factor regulated spatio-temporally during haematopoietic or cardiac development (Behrens et al., 2013) and the aim was to examine if controlled supplemental expression of HOXA9 resulted in more efficient production of mature cells.” It is better to present RTqPCR result using a ∆Ct method for Fig. 4 as fold changes are confusing especially in the context of transgene expression (the biological relevance of fold change is unclear unless one knows the base line transcript levels before gene activation).We believe that the ∆∆Ct method gives a better visualisation of the changes in transgene expression. The data in Figure 4 is presented as relative expression compared to untargeted iPSCs. Nevertheless, graphical presentation of ∆Ct values are provided in Extended data (Figure 9 Delta Ct representation of HOXA9 expression in AAVS1-targeted hiPSCs). In Fig 6 - Western Blot (Extended data), to test the peptide cleavage of P2A and T2A, the authors state that P2A is less efficient than T2A, as NpHR expression (following a P2A) was barely visible. The authors should provide positive control to exclude that the antibody for NpHR did not work.While the authors agree that this could enhance the data, our initial assessment was based on the 60kDa protein being, in all likelihood, a fusion of ChR2 (30kDa) and NpHR (30kDa). The anti-eNpHR antibody (catalog #AS12 1851; Agrisera) has been validated for use in Western Blot applications but unfortunately is not provided with a positive control sample. Southern blots should be performed to make sure that clones tested in this study were targeted in the corrected locus, and silencing was not due to random integration.The authors agree that integration of the cassette into the AAVS1 locus is vital. This was tested using PCR genotyping from outside the arms of homology and into the cassette as shown in Figure 1. As an alternative to southern blots, we have now included original PCR screening gels as extended data (Figure 7 – PCR screening of hiPSC clones to check for AAVS1 integration). In addition, we have included details of the sgRNA design (Figure 8 – sgRNA design) and show that all off-target locations occur in introns (Table 2.1 Guide 2 off-targets and Table 2.2 Guide 3 off-targets). ReferencesBehrens AN, Iacovino M, Lohr JL, et al.: Nkx2-5 mediates differential cardiac differentiation through interaction with Hoxa10. Stem Cells Dev. 2013;22(15):2211-2220. 23477547 10.1089/scd.2012.0611Klatt D, Cheng E, Hoffmann D, et al.: Differential Transgene Silencing of Myeloid-Specific Promoters in the AAVS1 Safe Harbor Locus of Induced Pluripotent Stem Cell-Derived Myeloid Cells. Hum Gene Ther. 2020;31(3-4):199-210. 31773990 10.1089/hum.2019.194Kondrashov A, Duc Hoang M, Smith JGW, et al.: Simplified Footprint-Free Cas9/CRISPR Editing of Cardiac-Associated Genes in Human Pluripotent Stem Cells. Stem Cells Development. 2018;27(6):391-404. 29402189 10.1089/scd.2017.0268 5882176Lopes LR, Zekaviti A, Syrris P, et al.: Genetic Complexity in Hypertrophic Cardiomyopathy Revealed by High Throughput Sequencing. J Med Genet. 2013;50(4):228-39. 23396983 10.1136/jmedgenet-2012-101270Luo Y, Liu C, Cerbini T, et al.: Stable enhanced green fluorescent protein expression after differentiation and transplantation of reporter human induced pluripotent stem cells generated by AAVS1 transcription activator-like effector nucleases. Stem Cells Transl Med. 2014;3(7):821–35. 24833591 10.5966/sctm.2013-0212 4073825Mosqueira D, Mannhardt I, Bhagwan JR, et al.: CRISPR/Cas9 editing in human pluripotent stem cell-cardiomyocytes highlights arrhythmias, hypocontractility, and energy depletion as potential therapeutic targets for hypertrophic cardiomyopathy. Eur Heart J. 2018;39(43):3879–3892. 29741611 10.1093/eurheartj/ehy249 6234851Ordovás L, Boon R, Pistoni M, et al.: Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition. Stem Cell Reports. 2015;5(5):918–31. 26455413 10.1016/j.stemcr.2015.09.004 4649136Smith JGW, Owen T, Bhagwan JR, et al.: Isogenic Pairs of hiPSC-CMs with Hypertrophic Cardiomyopathy/LVNC-Associated ACTC1 E99K Mutation Unveil Differential Functional Deficits. Stem Cell Reports. 2018;11(5):1226–1243. 30392975 10.1016/j.stemcr.2018.10.006 6235010" } ] } ]	1	https://f1000research.com/articles/8-1911
https://f1000research.com/articles/9-690/v1	09 Jul 20	{ "type": "Research Article", "title": "Impact of COVID-19 on the access to hearing health care services for children with cochlear implants: a survey of parents", "authors": [ "Mohammed Ayas", "Ahmad Mohd Haider Ali Al Amadi", "Duaa Khaled", "Ahmad Munzer Alwaa", "Ahmad Mohd Haider Ali Al Amadi", "Duaa Khaled", "Ahmad Munzer Alwaa" ], "abstract": "Background: The COVID-19 pandemic has affected the world in an unprecedented manner. It has aggravated psychological distress in parents of children with cochlear implants. Continuous use of a speech sound processor is critical for auditory stimulation in children with cochlear implants. However, movement restrictions imposed have affected access to hearing healthcare services. The current study explores the impact of the COVID-19 pandemic on hearing healthcare access for children with cochlear implants. Methods: An online questionnaire survey was conducted among parents of children with cochlear implants. Results: A total of 24 parents responded to the questionnaire. All the respondents reported that COVID-19 has a significant impact on access to hearing health services for their children. Speech processor breakdown and disconnection from the auditory mode of communication had a critical influence on behavioral changes in children. Conclusions: The current study highlights the hurdles faced by the parents in order to access hearing health services for their children. The use of innovative methods such as remote tele-audiology will be the way forward to tackle challenges faced by the parents.", "keywords": [ "Pediatric hearing loss", "Cochlear Implants", "Hearing health services", "Parental reactions" ], "content": "Introduction\n\nHearing is one of the most important senses in humans. Auditory development during a young age is critical for the acquisition of normal speech and language development1. Pediatric hearing loss constitutes one of the most important public health challenges2. Children with hearing loss are identified early and habilitated via hearing aids or with cochlear implants (CIs). Continuous auditory stimulation without any interruptions is essential for the successful attainment of language acquisition. Appropriate care and maintenance as well as continuous auditory verbal therapy (AVT) are also essential in attaining these goals3. These are managed by providing seamless access to hearing health services without interruption and restrictions.\n\nHowever, during the past six months, from the beginning of the year 2020, the world has witnessed an unprecedented attack on humans by a novel virus. The virus was proven to cause acute respiratory disease; the virus was later named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)4 and the disease coronavirus disease 2019 (COVID-2019). Towards the end of January 2020, the World Health Organization announced COVID-19 to be a public health emergency of great concern. This was followed by the announcement of stay at home orders and various precautionary measures enforced by various health authorities in order to contain the spread of the virus.\n\nThough the pandemic has had a profound psychological impact on all5, pediatric patients with CIs require additional attention to keep up with their communication needs6. Children with hearing loss pose significant challenges to their parents, particularly when there is limited access to their hearing care providers. The break in the routine of their hearing and therapy follow-up services has had considerable effects on the children as well as their parents7. Hence, the current study aims to explore the impact of the COVID-19 pandemic on hearing healthcare services for children with CIs.\n\n\nMethods\n\nEthical approval was obtained for the current study from the Ethics and Research committee of the University Hospital Sharjah (UHS-HERC- 034-20052020). All participants were contacted via telephone and verbal consent was obtained for participation in the study and publication of data. Owing to the current pandemic situation, movement restrictions and considering the safety of the participants during the study period, telephonic consent was obtained after thoroughly explaining the consent script. Once the script had been read to the participant, the authors recorded the participant’s agreement or disagreement to consent on the script sheet. This was approved by the Ethics and Research committee of the University Hospital Sharjah (UHS-HERC- 034-20052020) after review of the consent script.\n\nA cross-sectional study design was employed for the current study. This study was carried out at the audiology unit of University Hospital Sharjah, United Arab Emirates, for a period of two months from May 2020 to June 2020.\n\nConvenience sampling strategy was used to recruit participants during the study period. The study was targeted towards parents of CI children residing in the region close to the study center. To establish contact with the relevant study participants, professionals such as audiologists, speech language pathologists, auditory verbal therapists (AVTs) and otolaryngologists were identified and contacted in the region. These professionals were asked to share the survey with the desired parents of CI children. They contacted the potential participants through text messages with a link to the survey on.\n\nA questionnaire was developed (see Extended data)8 to gather answers to the research questions after discussion and mutual consensus from the authors. The authors’ background includes the specialties: audiology, speech language pathology, AVT and otolaryngology. The questionnaire consists of ten questions and was hosted on SurveyMonkey. The nature of the questions was equally divided into two categories. Five questions were focused on the challenges expressed by the parents. The other five questions were focused on CI user related challenges.\n\nHowever, the two categories of questions were presented in the questionnaire in a random manner. The questionnaire was prepared in English and translated into an Arabic language version for better comprehension by the respondents. Finally, backward translation was done from Arabic to English to assess any discrepancies in the translation of the questions.\n\nAs part of the piloting and face validity testing process, the questionnaire link was shared via text message with five parents of CI children. These participants were randomly selected and the feedback messages obtained from the parents were analyzed by the authors. Minor revisions were made to questionnaire based on the parent’s feedback. Two questions had ambiguous phrases, which were modified and reformulated in the final version of the questionnaire, for better comprehension of the parents.\n\nThe questionnaire was self-administered in nature, with a completion duration of less than five minutes. All the questions were created and presented in simple language to the parents of CI children. Care was taken while formulating the questions to capture the parents’ current feelings during the pandemic without referring to their past experiences. This would have been detrimental to exploring their obstacles owing to the current pandemic situation. A Likert response scale was used, in which parents were given the following options: 1) Strongly disagree, 2) Disagree, 3) Neither agree nor disagree, 4) Agree, 5) Strongly agree. The survey link was shared with the parents via text message. The participation of the parents was completely on a voluntary basis.\n\nThe obtained categorical data was descriptively analyzed and expressed in percentages using Microsoft Excel (2019). For the ease of analysis, the response constructed with the five alternatives were modified into three categories: strongly agree and agree were grouped and coded as 1) agree; category 2) was neither agree nor disagree; and options disagree and strongly disagree were categorized as 3) disagree. Finally, the analyzed data was presented in tables and graphs.\n\n\nResults\n\nA total of 31 parents of CI children were initially approached for the study. Out of these, 24 parents responded to the questionnaire sent to them (Table 1). All the CI users had pre-lingual deafness and the CI had been implanted for longer than one year at the time of the participation in the study.\n\nOf the questions relating to the parents, all of the parents (100%) reported that the COVID-19 pandemic has had an impact on availing timely hearing healthcare services for their children8. 96% of the parents reported that they could not follow up with their CI mapping dates with their centers. It was interesting to note that 88% of the parents felt that the COVID-19 pandemic has been psychologically distressing for them. However, 8% of them neither agreed nor disagreed with that statement, with 4% reporting that they totally disagree that psychological distress was caused by the pandemic. With respect to the home training programs and remote learning aspects, 96% of the parents agreed that the home training methods were challenging and 71% of the parents expressed that remote learning lessons were difficult for their children (Table 2, Figure 1).\n\nChallenges pertaining to the CI users were also reported and seemed to be drastically affecting the parents. With respect to the speech processor breakdown, 79% of the parents agreed that it affected the auditory communication with the child, whilst 17% disagreed. Interestingly, daily usage of the speech processor during the daytime was reported to be neutral by 50%, and 29% agreed that they used the speech processor adequately. However, regarding the access to auditory training sessions, 96% of the parents reported that they had difficulty in accessing the services. 67% agreed that behavioral changes were common during the stay at home restriction period, with 8% disagreeing with this. The home training programs provided by their clinicians or AVTs were reported to have been followed accurately by 50% of the parents. However, significant number of parents reported that they did not follow the methods at home (Table 3, Figure 2).\n\n\nDiscussion\n\nThe current study aims to understand the impact of COVID-19 on access to hearing healthcare services for children with CI. The results of the study underline the fact that the current pandemic situation has significantly affected children with CI.\n\nIt is an undisputable fact that hearing healthcare access is a complex issue which involves an interplay of multiple factors from both parents as well as care providers2,7. Pediatric hearing loss has already proven to be a challenge for the parents during the pre – pandemic times. Parents of CI children enrolled in the current study reported that they faced issues with not receiving timely hearing health services for their children. These are mainly related to the break in CI mapping follow-up schedules and speech processor break down. It was reported extensively in the literature that for a successful outcome with CI, continuous usage of the speech processor is critical9–11. In addition, optimal current levels are necessary for the development of auditory sensation12,13. It is also evident from the current study that the present situation is psychologically pressing for both parents as well as for their children.\n\nParents have also reported that home training activities were difficult. This could be attributed to the boredom faced by both children and parents due to being confined at home for a long duration. In such scenarios, the child may not be able to cooperate with the task to achieve the target goal assigned by their clinician. Moreover, they faced greater trouble adapting to the remote learning environment.\n\nIt also worth noting that CI user related issues caused an additional burden on the parents. For example, in the current study, 79% of the parents reported that speech processor breakdown affected their auditory mode of communication with the child. It is also important to consider that not all CI users will have spare CI processors readily available at home for replacement14. This will add to the psychological anxiety of both parents and their children. It is documented in literature that a breakdown of the CI processor or temporary discontinuation of speech processor usage is strongly correlated with expected outcomes in CI children7. Interestingly, our results suggest that in the current situation, significant behavioral changes in CI children are shown. This can also be attributed to the lack of access to an auditory mode of communication15,16. Such global changes in behavior and emotional aspects of parents as well as children will have severe impacts on following the home training programs planned by their clinicians.\n\nThe findings from our study focused on the enhanced need for the timely access to hearing health services for CI children. The study also shed light on the various needs and hurdles faced by parents. One of the critical aspects of improving service accessibility is reaching out to users through innovative methods. This includes tele-audiology services17–19, in which the healthcare professionals can provide remote mapping and necessary trouble shooting for the speech processors. Also, a tele-AVT program is another option for parents to engage the child with therapists. This will ensure better compliance and continuity of the care plan. In addition, this will facilitate containment of behavioral changes in children to a certain extent.\n\nDuring these unprecedented times, hearing health services can also be availed via homecare visits or mobile hearing health services. However, such services are not widely available. In addition, precautionary measures are critical for both children and healthcare professionals before organizing the home visits.\n\n\nConclusions\n\nThe COVID-19 pandemic has been reported to have a considerable impact on access to the hearing health services availed by CI children. Parents and CI children are distressed due to the lack of access to services and consequent breakdown in communication. Results from the current study suggest the need for more innovative methods to be employed to meet the needs of CI children. However, the current study has a limitation due to the restricted sample size of participants. Future studies can be carried out by focusing on a wider CI population and their specific needs.\n\n\nData availability\n\nFigshare: Impact of COVID-19 on the access to hearing health care services for children with cochlear implants: a survey of parents. https://doi.org/10.6084/m9.figshare.12503510.v38\n\nThis project contains the following underlying data:\n\nAyas data.xlsx (Questionnaire data in Microsoft Excel format)\n\nFigshare: Impact of COVID-19 on the access to hearing health care services for children with cochlear implants: a survey of parents. https://doi.org/10.6084/m9.figshare.12503510.v3 8\n\nThis project contains the following extended data:\n\nExtended data-Questionnaire.docx\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC BY 4.0).", "appendix": "Acknowledgments\n\nThe authors would like to thank all the parents who participated in the online survey.\n\n\nReferences\n\nVan Den Abbeele T, Crozat-Teissier N, Noel-Petroff N, et al.: Neural plasticity of the auditory pathway after cochlear implantation in children. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWheeler A, Archbold S, Hardie T, et al.: Children with cochlear implants: The communication journey. Cochlear Implants Int. 2009; 10(1): 41–62. PubMed Abstract \| Publisher Full Text\n\nGeers A: Factors Affecting the Development of Speech, Language, and Literacy in Children With Early Cochlear Implantation. Lang Speech Hear Serv Sch. 2002; 33(3): 172–183. PubMed Abstract \| Publisher Full Text\n\nAyas M: Impact of COVID-19 on the access to hearing health care services for children with cochlear implants: a survey of parents. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12503510.v3\n\nGeers A, Nicholas J, Tobey E, et al.: Persistent Language Delay Versus Late Language Emergence in Children With Early Cochlear Implantation. J Speech Lang Hear Res. 2016; 59(1): 155–170. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchafer E, Thibodeau L: Speech Recognition in Noise in Children With Cochlear Implants While Listening in Bilateral, Bimodal, and FM-System Arrangements. Am J Audiol. 2006; 15(2): 114–126. PubMed Abstract \| Publisher Full Text\n\nTeagle HFB, Park LR, Brown KD, et al.: Pediatric cochlear implantation: A quarter century in review. Cochlear Implants Int. 2019; 20(6): 288–298. PubMed Abstract \| Publisher Full Text\n\nGagnon EB, Eskridge H, Brown KD: Pediatric cochlear implant wear time and early language development. Cochlear Implants Int. 2020; 21(2): 92–97. PubMed Abstract \| Publisher Full Text\n\nLiu S, Wang F, Chen P, et al.: Assessment of outcomes of hearing and speech rehabilitation in children with cochlear implantation. J Otol. 2019; 14(2): 57–62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSilverman CA, Schoepflin JR, Linstrom CJ, et al.: Repair Issues Associated With Cochlear Implants in Children. Otol Neurotol. 2010; 31(6): 926–931. PubMed Abstract \| Publisher Full Text\n\nKhan S, Edwards L, Langdon D: The Cognition and Behaviour of Children with Cochlear Implants, Children with Hearing Aids and Their Hearing Peers: A Comparison. Audiol Neurootol. 2005; 10(2): 117–126. PubMed Abstract \| Publisher Full Text\n\nStevenson J, Pimperton H, Kreppner J, et al.: Emotional and behaviour difficulties in teenagers with permanent childhood hearing loss. Int J Pediatr Otorhinolaryngol. 2017; 101: 186–195. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwanepoel DW, Hall JW: A Systematic Review of Telehealth Applications in Audiology. Telemed J E Health. 2010; 16(2): 181–200. Aiello, C. P., & Ferrari, D. V. (2015). Teleaudiology: efficacy assessment of an online social network as a support tool for parents of children candidates for cochlear implant. Language,.15(9.3), 13-4. PubMed Abstract \| Publisher Full Text\n\nSharma SD, Cushing SL, Papsin BC, et al.: Hearing and speech benefits of cochlear implantation in children: A review of the literature. Int J Pediatr Otorhinolaryngol. 2020; 133: 109984. Buckman, M. and Fitzharris, K., 2020. Teleaudiology and Cochlear Implant Appointments. The Hearing Journal,. 73(5), p.30. PubMed Abstract \| Publisher Full Text\n\nBuckman M, Fitzharris K: Teleaudiology and Cochlear Implant Appointments. Hear J. 2020; 73(5): 30. Publisher Full Text" }	[ { "id": "66847", "date": "16 Jul 2020", "name": "Rohit Ravi", "expertise": [ "Reviewer Expertise Pediatric Audiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe COVID-19 pandemic has hindered access to hearing healthcare. The present study highlights the impact of the same on children with CI. The survey is the need of the hour to find out the challenges and the ways in which this can be adopted to overcome these challenges.\nI appreciate the authors for undertaking this relevant topic, the study is well written and will definitely be beneficial to the readers & scientific community.\nThere are a few minor changes I suggest:\nTitle can be modified to include the location of the study as \"Sharjah\".\n\nAlso, add the limitation and clinical implication of the study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "66848", "date": "22 Jul 2020", "name": "Holly S. Kaplan", "expertise": [ "Reviewer Expertise Audiology." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI found the reference list to be timely and provides citations to give evidence to statements given. Older references would be viewed as more seminal in the field or were written by known experts. I would remove the sentence in the abstract that states parents are experiencing aggravated psychological distress. Your paper found that to be true based on your sample, but I do not believe there are other papers out already that have found the same issues. I would re-word that psychological distress may be a consequence.\n\nThe study design is appropriate and the questionnaire includes questions that pair to demonstrate consistency in the study. This study could be easily replicated. It would be interesting to do so in different countries or in one country with differing access to CI care. I would also want to re-do this study with the same group post covid. Do the questions on daily use change?\n\nI noted 'partly' on the reproducibility as the reader must go to another link to see the actual questions and the data. I would recommend giving each question a key word title, e.g., Q1 could be called Q1Access and Q2Map. This would make reading the tables easier and clearer.\n\nI would also look into further statistical analysis of the data. Are there significant differences in and between question responses? An analysis to determine which questions actually match to each other would also be interesting. Length of implant use, age of child, number of children in family etc. may also impact the answers. I noted you felt the N was low in your group, but I commend you in finding this many parents who are willing to complete the survey during these difficult times.\n\nThe source data is available, but the reader must of course go to the website where it is located. The conclusions are conservative and reflect the data. I appreciate the difficulty experienced by these families and their honesty in noting AR therapy is hard to keep up with when there are so many things changing rapidly.\n\nMy one concern would be behavior changes ascribed to implant use -- not sure that your question about behavioral changes is necessarily tied to implant use. As an anecdote, most of my friends are commenting on their children's behavior changes as a result of lockdown and social distancing.\n\nI commend the authors in getting this study completed and published so quickly and under pandemic conditions. Excellent work that can be expanded on!\n\nThe following comments are from Dr. Morrison.\n\nReviewer comments:\nThe authors are applauded for executing this study during difficult times when recruitment and engagement with participants is challenging. This article is recommended for publication with minor revision. The following comments are provided:\n\nThe authors indicate that “Care was taken while formulating the questions to capture the parents’ current feelings during the pandemic without referring to their past experiences”. Were additional instructions (not printed on the questionnaire) provided to parents? Several of the questions seem quite general in nature and difficult to directly connect to the experience of parenting a child with CI during the pandemic. For example, the authors note that 88% of the parents report that COVID-19 is psychologically distressing for the parent and in the conclusion state that “parents and CI children are distressed due to the lack of access to services and consequent breakdown in communication.” However, the formulation of this question - at least in English - does not necessarily relate the psychological distress to CI issues or access to hearing healthcare but could be due to their own safety, fear for elderly parents, stress related to work or finances, etc. Additional explanation for how the parents were instructed to answer these questions as specifically related to CI would be helpful. If this information was in the telephone script, the inclusion of the script in the appendices should be considered. Alternatively, if the question was intended to probe psychological distress as a whole, rather than related to hearing healthcare, the authors are encouraged to discuss the role of general parent distress on development of the hearing-impaired child.\n\nAdditionally, question #4 probed whether speech processor use at home was “adequate” – for which 29% of the parents agreed and 50% of parents neither agreed nor disagreed. Elaboration as to whether speech processor use at home was believed to be different during this pandemic time period compared to typical home use would be helpful. The authors describe that 79% of parents reported that speech processor breakdown affected auditory communication with the child. The authors are encouraged to elaborate whether speech processor breakdown was more frequent or more prolonged during this pandemic time. The authors are encouraged to describe the process for replacing/repairing the speech processor in the UAE (i.e. does this require a trip to a hearing healthcare center? – in the reviewer’s home country, it does not). The authors are encouraged to describe in additional detail the current average delay for hearing healthcare services in the study area (i.e. are most appointments delayed by 1-2 weeks, are all hearing-related appointments cancelled indefinitely, etc.) to provide additional context for the international reader.\n\nLastly, question 6 asked parents to reflect on behavioral changes in children with CI in the home. The authors note that “in the current situation, significant behavioral changes in CI children are shown. This can also be attributed to the lack of access to an auditory mode of communication.” The authors are encouraged to describe whether additional instruction existed to inform the parents to reflect on behavior changes specifically related to communication (vs boredom or disruption of routines, for example) or whether they are hypothesizing the link between the two.\n\nIs the work 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": [] } ]	1	https://f1000research.com/articles/9-690
https://f1000research.com/articles/9-684/v1	07 Jul 20	{ "type": "Research Article", "title": "Venezuelan migrant population in Colombia: health indicators in the context of the Sustainable Development Goals", "authors": [ "Laura Juliana Bonilla-Tinoco", "Melissa Aguirre-Lemus", "Julián Alfredo Fernández-Niño", "Laura Juliana Bonilla-Tinoco", "Julián Alfredo Fernández-Niño" ], "abstract": "Background: The number of Venezuelan migrants in Colombia has dramatically increased over the past years, which poses great challenges to the Colombian health system. Therefore, the aim of this study was to compare some health indicators related to the Sustainable Development Goals between the Venezuelan migrant population and the Colombian population. Methods: A longitudinal, descriptive analysis of the maternal mortality ratio; the neonatal, infant and under-five mortality; the proportionate mortality due to undernourishment; and the rates of alleged sexual felony, intimate partner violence and domestic violence in the Venezuelan migrant population in Colombia and in the Colombian population in the 2015-2019 period was conducted. Maternal and child health and undernourishment indicators were estimated for the 2015-18 period, while the gender-based violence indicators were obtained only for 2018-19, since those were the years with information available for each of these indicators. Data was extracted from official sources, such as the National Administrative Department of Statistics (DANE), National Institute of Legal Medicine and Forensic Sciences (INMLCF) and Migración Colombia. The categorical and numerical variables were described through percentages and rates, respectively. Results: Venezuelan migrants in Colombia had higher rates of maternal, neonatal, infant and under-five mortality, as well as proportionate mortality due to undernourishment, than the Colombian population throughout the study years, although the difference between them decreased at the end of the period. As for the gender-based violence indicators, the Colombian population showed higher rates than the Venezuelan migrants, and both Colombian and Venezuelan female victims showed higher rates in these violence indicators than their male counterparts of the same nationality. Conclusions: Some apparent inequalities still persist despite the efforts of the Colombian government to attend to the health needs of the Venezuelan migrant population. Colombia must keep and strengthen migratory inclusion in its public policies to impact on migrants’ health.", "keywords": [ "Transients and Migrants", "Emigration and Immigration", "Health Status Indicators", "Colombia", "Venezuela" ], "content": "Introduction\n\nPrior to recent years, Colombia had never experienced a massive immigratory wave in its whole history as a Republic. Contrarily, this country has registered three large emigration waves during the last century, in which many Colombian people migrated to the United States, Venezuela and Spain in the 60’s, 80’s and 90’s, respectively. Consequently, Colombia has been historically considered an expelling country and had almost no experience as a recipient of international migrants1.\n\nNonetheless, the number of Venezuelan migrants in Colombia has been increasing since the early 2000’s, inasmuch as high-income Venezuelans progressively began to migrate to Colombia since 2005 as a consequence of the political and socioeconomic crisis in Venezuela. Furthermore, with the worsening of the crisis in 2014, Colombia started to experience new modalities of migratory movements from Venezuela, specifically from the Táchira, Carabobo, Zulia, Barinas and Lara states. The initial flow included mainly a large proportion of deported and returned Colombians, as well as of binational citizens and their families, but then began to be mainly constituted by migrants of irregular condition, including refugees, pendular migrants, migrants in transit and irregular migrants that intend to stay in Colombia2. Additionally, the migratory phenomenon from Venezuela has been dramatically growing, reaching a massive peak in 2017–20183, so that as of December 2019, roughly 1.7 million Venezuelan immigrants resided in Colombia. Bogotá D.C, Norte de Santander, Atlántico, La Guajira and Antioquia are the zones of highest concentration, with over 100,000 Venezuelan migrants reported in each zone4. Moreover, by that date only 754,085 were regular migrants, while the remaining majority (1,017,152) had an irregular migratory status. The latter indicates that, currently, more than half of the Venezuelan migrants in Colombia (57.43%) do not have any type of Colombian documentation that would allow them access to health insurance and formal jobs in Colombia4.\n\nHealth status and healthcare access for Venezuelans have been considered big challenges for the Colombian government from the very beginning of the migratory crisis2, considering that an increase in cases of public health interest events has been reported among migrants from Venezuela in Colombia, as indicated in the “27th Bulletin - Notification of public health interest events during the migratory phenomena”5. The events with the highest reported cases among people coming from Venezuela were malaria, gestational syphilis, domestic and gender based violence, extreme maternal morbidity, HIV/AIDS mortality and acute undernourishment in under-fives5.\n\nGiven the growing dynamics of the Venezuelan migratory phenomenon, its impacts on the health sector and the challenges it represents, the Colombian government has generated a quick response to attend to the health problems and needs of the migrant population. Thus, the “Response Plan to the Migratory Phenomena from the Health Sector and the document of public policy by the National Council for Economic and Social Policy # 3950” (Conpes 3950 in Spanish) was designed6. The first document enabled health services access for regular migrants, indigenous people and Colombian returnees, and comprehensive care for pregnant women and children less than a year old, independently of their migratory status; while irregular migrants can only access emergency services and nationwide public health interventions1. The second document determined strategies and lines of action to identify needs in the supply of health services; provide technical assistance to increase health insurance for regular migrants and Colombian returnees; and promote monitoring of the irregular migrants’ health care6.\n\nConsidering all of the above, the analysis of the migratory phenomenon at the national level must include the characterization and health diagnosis of the Venezuelan migrant population in Colombia. In addition, it is necessary to evaluate health outcomes inequalities between Colombians and Venezuelans in Colombia as a potential and emergent issue. This assessment should be done taking into account the 2030 Agenda for Sustainable Development, which was set up by the World Sustainable Development Summit (WSDS) in 2015 in New York and describes the 17 Sustainable Development Goals (SDG) and their 169 universal targets that are valid for 15 years (as described by the International Organization for Migration). These SDGs are shared between countries and used to monitor progress under an equality perspective, so each country has to develop effective strategies and mechanisms to achieve the SDG according to their social needs.\n\nAdditionally, the SDGs not only have health-related objectives (maternal and child health, malnutrition and gender violence), but also consider migration to be an essential topic to contribute to the development on each country. This is stated in the 10.7 target from the 10th objective, which appeals for “orderly, safe, regular and responsible migration and mobility of people” and the design of well-managed migration policies7. Based on this, the comparison of health results between Venezuelan migrants and Colombian people allows us to ascertain the presence of gaps in the inclusion of the migrant population in the host population and the degree of fulfillment of the SDGs in Colombia. Thus, the objective of this study was to compare some key health indicators, related to the SDGs, between the Venezuelan migrant population in Colombia and the Colombian population.\n\n\nMethods\n\nThe current investigation is a longitudinal, descriptive analysis of some health indicators related to maternal and child health, undernourishment and gender-based violence in the Venezuelan migrant population in Colombia and in the Colombian population in the 2015–2019 period. The analyzed health indicators were chosen based on their relation to the second (zero hunger), third (good health and well-being) and fifth (gender equality) SDGs.\n\nThe study was conducted using official records from the following governmental institutions: National Administrative Department of Statistics (DANE, from its initials in Spanish), National Institute of Legal Medicine and Forensic Sciences (INMLCF, from its initials in Spanish) and Migración Colombia, which is the Colombian migration agency. These records are publicly accessible on the institutes’ websites and can be downloaded without any formal request.\n\nData from the DANE were taken from the Vital Statistics section, which is based on live birth and death certificates, and on the certificates issued by the Civil Registry officials when there has not been contact with a health professional. Information from this source allowed the construction of the following indicators: maternal mortality ratio, neonatal mortality, infant mortality, under-five mortality, and proportionate mortality due to undernourishment. Data extraction was made from the files corresponding to the years 2015–18; information from 2019 has not been published yet. In these files, nationality was ascertained through a question regarding the country of regular residence of the deceased person or of the mother of the newborn (for deaths in children less than a year and for the live births) when they resided in a country that was not Colombia.\n\nData from the INMLCF include all external cause of injury cases that occurred in any site of the Colombian territory, have a technical report and are directly or indirectly known by the INMLCF8. In the case of Migración Colombia, the estimated figures are based on the information obtained through the assessment and cross-referencing of administrative registries, such as the Special Residence Permit (PEP, from its initials in Spanish); the Administrative Registry of Venezuelan Migrants (RAMV, from its initials in Spanish); the Foreign Registry Information System (SIRE, from its initials in Spanish); migratory entries (hosting intention); and estimations of irregular migrants made by the migratory authority4,9. Furthermore, the population projections made by the DANE for the 2018–2023 period were also used; these are calculated based on the results from the National Population and Housing Census from 2018.\n\nThese three sources allowed the estimation of alleged sexual felony, intimate partner violence and domestic violence rates. The INMLCF makes a distinction between intimate partner violence and domestic violence in that the latter regards a wider type of violence that comprises violence against children, intimate partner violence, violence among other family members and violence against the elderly. Nonetheless, the indicators were estimated just for 2018 and 2019 because the victim’s nationality started being registered in the INMLCF records as of 2018 and because public data from Migración Colombia regarding the number of Venezuelan people in the Colombian territory were more complete and specific as from this same year. It is worth clarifying that although information about non-fatal injuries of external cause for 2019 has a cut-off date of December 31st, it is still labeled as preliminary. Similarly, the latest 2019 infographic released by Migración Colombia was published in early 2020. The indicators for the Venezuelan population used data from Migración Colombia as denominators, while indicators for Colombian people used information from the DANE population projections. In both cases, the numerator was extracted from the INMLCF files.\n\nMaternal mortality ratio (MMR): refers to the ratio between the number of maternal deaths and the number of live births for a given time and space (expressed per 100,000 live births). According to the Pan-American Health Organization (PAHO), a maternal death refers to the death of any woman who died while pregnant or in the 42 days following the pregnancy ending, irrespective of the site and duration of the pregnancy, and whose cause of death was related or worsened either by the pregnancy or the care of it. The latter entails having an International Classification of Diseases, 10th revision (ICD-10) diagnosis of O00-O99 (except for O96 and O97) in the basic cause of death10. Based on the above, we included all records of women whose basic cause of death was any of the indicated ICD-10 diagnoses, who were of reproductive age (10–59 years for the present study) and who had a positive answer either to the question asking whether the woman was pregnant the moment she died or had been pregnant in the six weeks prior to the death.\n\nNeonatal, infant and under-five mortality: all indicators use the number of live births (LB) as denominators, whereas the number of deaths in newborns aged 0–27 days; in children aged less than a year; and in children aged less than five years were used as the numerators for the neonatal, infant and under-five mortality, respectively10. Data extraction involved filtering by the age of the deceased person, so that all records that fulfilled the age criteria were included, irrespective of the diagnosis of the basic cause of death. The results are expressed per 1,000 LB.\n\nProportionate mortality due to undernourishment: was estimated as the percentage of deaths that occurred in each year of the study period due to undernourishment (number of deaths due to undernourishment divided by the total deaths per year). A death due to undernourishment was considered to be a death that had an ICD-10 diagnosis of E40-E46, E50-E64, D513, D520 or D5 in the basic cause of death; the selected diagnoses are based on the “Nutritional deficiencies and nutritional anemias” group from the PAHO’s 6/67 mortality list11. Additionally, the age distribution of the deaths due to undernourishment was also assessed by dividing the number of undernourishment deaths in each age group of interest (less than a year, under five years and adults 65+ years) into the total number of undernourishment deaths per year.\n\nRates of alleged sexual felony, intimate partner violence and domestic violence: the number of cases of alleged sexual felony, intimate partner violence and domestic violence served as numerators, while the total population of Venezuelan people in Colombia (extracted from the Migración Colombia infographics) and the total Colombian population (population projections estimated by the DANE) served as the denominators for the Venezuelan and the Colombian indicators, respectively. Rates by sex were estimated only for 2018 because the information of the 2019 infographic was not disaggregated by sex. The results are reported per 100,000 habitants.\n\nA descriptive analysis was conducted by means of numerical and graphical methods. The categorical and numerical variables were described through percentages and rates, respectively; and trend, bar and stacked bar charts were used to graph the estimates. All indicators were estimated using Microsoft Excel 2013 and graphics were done in Tableau 2019.2.1 for visualization purposes; however, graphics can also be constructed in Microsoft Excel.\n\nThe present study was based on administrative, secondary information, i.e. official national records, which are properly anonymized, preventing individual identification; are of public access; and do not contain personal information. Given the above and that it was not conducted on human participants, this investigation did not need approval of an ethics committee, according to the 8430 Resolution of 199312,13.\n\n\nResults\n\nTable 1 shows all the variables needed to estimate the health indicators. Broadly, maternal mortality ratio and neonatal, infant and under-five mortality (Figure 1 and Figure 2) were found to be higher in Venezuelans than in Colombians throughout the whole study period, even reaching values higher than the SDG 3 targets for maternal (<70 maternal deaths per 100.000 LB), neonatal (<12 neonatal deaths per 1,000 LB) and under-five mortality (<25 neonatal deaths per 1,000 LB). Nonetheless, these indicators also displayed a downward trend, even accomplishing the goals for neonatal mortality at some points (2017–18). On the other hand, trends of the indicators for Colombians were stable throughout the study years and their values were always within the expected goals.\n\nThis table presents the raw data necessary to estimate the health indicators analyzed in this study.\n\nLatest vital statistics data (live birth and deaths) is from 2018.\n\nInformation about violence (alleged sexual felony, intimate partner violence and domestic violence) disaggregated by nationality was available only as of 2018.\n\nSex-disaggregated information about the total Venezuelan population in Colombia was available only for 2018.\n\nThe maternal mortality ratio trend was stable in the Colombian population throughout the study period, while it showed a downward trend in the Venezuelan migrants. Additionally, the Venezuelan migrants had notably higher figures than the Colombian population.\n\nLB: live births.\n\nRatios are reported per 100,000 LB.\n\nThe mortality figures were always lower in the Colombian population than in the Venezuelan migrants, although figures for the latter had a downward trend during the study years, so that the difference in the mortality indicators between populations was smaller at the end of the period.\n\nLB: live births.\n\nRates are reported per 1,000 LB.\n\nRegarding proportionate mortality due to undernourishment, the Colombian population had a stable trend during the study period (0.8% in 2015–16 and 0.7% in 2017–18), while the Venezuelan population showed increasing values over time (2.6%, 1.5%, 3.2%, and 5.9% in 2015, 2016, 2017 and 2018, respectively). It can be seen that even the lowest value for the Venezuelan population is roughly two-fold the highest value of the Colombian population. Additionally, the age distribution of proportionate mortality due to undernourishment (Figure 3) showed a different epidemiological profile of affected people: whilst deaths due to undernourishment predominantly affected adults 65+ in Colombians (over 60% in each year of the study period), children were the most affected in Venezuelans (accounting for more than 75% of undernourishment deaths in 2016–18).\n\nThe age distribution of the undernourishment deaths clearly show a different profile of affected groups: while older adults are the most affected in the Colombian population, children represent the majority of those who died from undernourishment in the Venezuelan migrant population.\n\nEstimations were made by dividing the number of undernourishment deaths in each group of interest by the total number of undernourishment deaths per year. This means the reported percentages do not add up to 100% because only age groups of less than a year old, under-fives and adults aged 65+ were assessed.\n\nAs for violence, the Colombian population had higher rates of all types of violence (alleged sexual felony, intimate partner violence and domestic violence) in both 2018 and 2019. However, the Colombian population had a slight decrease in its violence rates from 2018 to 2019, while the contrary occurred in the Venezuelan population, resulting in less pronounced differences between the two population’s rates. In addition, domestic violence was the most frequent type of violence in both Colombians and Venezuelans (Figure 4). On the other hand, sex disaggregation revealed that, in 2018, female victims had higher rates in all of the gender-based violence indicators in comparison to male victims, and that Colombian women had higher violence rates than Venezuelan women. Once again, domestic violence was the most frequent type of violence in both Colombian and Venezuelan men and women (Figure 5).\n\nThe Colombian population had higher violence rates than the Venezuelan migrants in all types of violence in both years. However, the violence rates increased in the Venezuelan migrant population in 2019, so the differences between populations decreased from one year to another. In both years and population groups, domestic violence was the most frequent type of violence.\n\nData from 2019 is still preliminary. Cut date: 31-12-2019.\n\nTotal rates are reported per 100,000 habitants.\n\nDomestic violence includes violence against children, intimate partner violence, violence among other family members and violence against the elderly.\n\nIt can be seen that both Colombian males and females showed higher violence rates than the Venezuelan migrants, but women were always more affected than men in every analyzed type of violence, so that violence rates were highest among Colombian female victims. As previously noted, domestic violence was the most frequent type of violence among women and men, and in both Colombians and Venezuelan migrants.\n\nTotal rates are reported per 100,000 habitants.\n\nDomestic violence includes violence against children, intimate partner violence, violence among other family members and violence against the elderly.\n\n\nDiscussion\n\nCurrently, literature about Venezuelan migrants’ health status is limited; even Colombia lacks sufficient information on this subject despite being the main receptor of said migratory phenomenon, though some research has been done14. Hence, the present study aimed to make a comparison of some SDG-related health indicators between the Venezuelan migrants in Colombia and the Colombian population, which showed that maternal and child and undernourishment indicators in the former were always worse than in the latter during the study period (although the gap between Colombians’ and Venezuelan migrants’ indicators tended to decrease throughout the years). Conversely, the Colombian population had higher violence rates than the Venezuelan migrants.\n\nSome other efforts to characterize the Venezuelan migrants in Colombia and their health needs have been made. For example, a report made by Profamilia describes the unmet sexual and reproductive health needs of the Venezuelan migrants in Colombia, and also presents figures for the Venezuelan country for some of the indicators analyzed in this study15, although they are likely to be inaccurate because the Venezuelan government has not produced official mortality statistics since 201316. Thus, the maternal mortality ratio in Venezuela in 2015 was reported to be 95 maternal deaths per 100,000 LB and the infant mortality in 2016 was 12.5 per 1,000 LB15. Another example is the “Migración Venezuela Project”, whose report on migrants’ use of health care services shows women and individuals aged 20–29 years were the ones who used health services the most. Furthermore, it indicates the principal causes for hospitalization were those related to pregnancy, delivery and puerperium, and that the number of legal-medical exams performed on victims of alleged sexual felony steadily increased from 2017–201917. Although that report does not address the same indicators presented in this study, its information is complementary to our estimations and helps in understanding the health needs of the migrant population.\n\nDespite all the efforts the Colombian government has made to attend the health needs of the Venezuelan migrants, there are still some apparent inequalities, as shown by the low accomplishment of the second and third SDGs in the Venezuelan migrant population, evidenced through the notable differences in the indicators related to maternal and child health and undernourishment. The above could be due to barriers to access health care services, which directly affects the migrants’ health outcomes. Considering Andersen’s Behavioral Model of Health Services Use and its subsequent revisions18, the policies designed to respond to the migratory phenomenon can be framed within the enabling factors because they enable and regulate health care for regular migrants, pregnant women and children aged less than a year, and prepare the territories and the strengthen the health system in response to the increasing health care demand by migrants. Additionally, migratory status is also an enabling factor, since an irregular migratory status serves as a barrier to use the health services not only because it grants access to just emergency services, hindering the appropriate monitoring of other health conditions, but also because these particular migrants might avoid using health services out of fear of being deported. At the same time, this fear of being forcefully returned to their country of origin can be considered a predisposing factor, given the status of these individuals in the community (social structure); sex, age and education are also predisposing factors. Furthermore, perceived health status and health conditions that clearly require medical attention (pregnancy, undernourishment, etc.) represent the need component of the model.\n\nAdditional to the difficulties in accessing health services, another possible explanation for the Venezuelan migrants having worse indicators might be the previous and chronic exposure they had to medicine, food and basic public services shortages, and to an undermined health system in their country of origin. Venezuela’s socioeconomic crisis has produced numerous public health issues, such as alarming increases in neonatal, infant and maternal mortality, undernourishment and vector-borne diseases16,19,20. Added to this vastly poor health situation, a great number of Venezuelan migrants who come to Colombia cross the boarders by walking, thereby walking long distances, exposing themselves to extreme temperatures and other multiple risks21, which can also negatively impact their health.\n\nOn the other hand, the apparent gap in the health indicators for the analyzed populations might be overestimated due to difficulties related to the data and its collection. First, there might be a low quality of the registered data before 2017, when two national directives, the external circulars 012 and 029, were given in order to systematize and improve the reporting of data of the health services given to foreigners in health facilities from border22 and non-border zones23. These two documents, which describe the process of sending a series of files from the health service facilities and indicating the nationality of the patient, helped to enhance information systems regarding the Venezuelan migrants’ health data that was available to characterize them. Second, there was also a low level of surveillance, registry and reporting of health events in the migrant population at the beginning of the migratory crisis, which could also have impacted the registered data. Third, data limitations can also be another factor, since estimates from Venezuelan migrants are more unstable due to their small denominators, which is clearly seen in the maternal mortality ratio results. Fourth, the apparent lower violence indicators in Venezuelan migrants might be a reflection of a higher reluctance to report a case of violence out of fear of being deported or for the reversal of the process against them. Another currently documented reason is the lack of empowerment and leadership of the institutions in charge of attending violence cases, which increases misinformation in the migrant population, who do not know where to go and which entities could provide support24–26. However, it should be noted that over the past two years, different sectors, such as health, protection, justice, education, and the public ministry, have been coordinating efforts to strengthen the route against gender-based violence in the territories, which varies according to the institutional capacity in each territory. Similarly, some Colombian departments have received technical support and accompaniment from international organizations and the health ministry in order to establish strategies that contribute to mitigate gender-based violence in the migrant population (such as the UN Women ‘Valiantes’ strategy, UN Women and National Planning Department workshops, the UN Women ‘Diálogos entre Lideresas colombianas y venezolanas’ initiative and the Ministry of Health’s ‘Comprehensive approach to gender violence’).\n\nConsidering the Venezuelan migrants’ characteristics, their health needs and the growing demand on health services, the public policy Conpes 39506 was designed as part of the Colombian government’s response to the migratory phenomenon. Its strategies and lines of action include: 1) identification of the needs of health services in territories that are affected by the Venezuelan migration to define the needs for strengthening the installed capacity in infrastructure, human talent and biomedical supplies of health facilities; 2) provision of technical assistance to increase the Venezuelan migrants’ and Colombian returnees’ affiliation to the health system, and to keep a follow-up of the health events of irregular migrants; 3) improvement of the public health response capacity of the host territories and communities, emphasizing the strengthening of the public health surveillance response capacity for a timely detection of public health interest events and the issuance of early alerts; and 4) establishment of migratory flexibility mechanisms for the incorporation of the Venezuelan migrant population. Additionally, different institutions, organizations and cooperatives have established alliances and articulated efforts to form a balanced offer of health care according to the needs of the population, implementing “Mobile Units” in the territories to support institutional capacity. These units allow the provision of advice, guidance, and health care to the Colombian and Venezuelan population, which promotes prioritization and attendance of events of public health interest and strengthening information and processes (for example, the Colombian Institute for Family Welfare (ICBF) and the United Nations Agency for Refugees (UNHCR) agreement to strengthen care for refugee and migrant children from Venezuela, the introduction of mobile justice units in Bogotá by the Ministry of Justice and Law, and the ‘Salud para la paz’ initiative).\n\nSome administrative difficulties in accessing health care persist despite the establishment and implementation of the lines of action of Conpes 3950, which relate to the Venezuelan migrants’ lack of knowledge and disinformation about the functioning of the Colombian health system and of the process to effectively affiliate to it. For example, although migrants who have the PEP have the chance to affiliate to the Colombian health system, a lot of them prefer to engage in other activities instead of completing the administrative procedures because they imply a time and money investment, which usually they cannot afford. Furthermore, it has been seen that even when a Venezuelan migrant affiliates to the Beneficiary Selection System for Social Programs (SISBEN, from its initials in Spanish), obstacles to access health care still persist27.\n\nBased on the above and on the Venezuelan migrants' panorama from the last five years, the Colombian government must keep and strengthen migratory inclusion in its public policies to achieve a direct and positive impact on the social determinants of health. Said restructuring would respond to the obligation of states to respect and guarantee the rights and freedoms recognized internationally to all people, regardless of their immigration status28; therefore, Colombia must take measures to balance, adapt and redistribute resources, in order to improve the health status of its residing population, regardless of their nationality. In addition, it must take into account universal health coverage, which emphasizes comprehensive access to all health services and equity in health without financial deprivation.\n\nOn the other hand, it also has to be kept in mind that the migratory phenomenon coming from Venezuela is classified as a South-South Migration, which is characterized by a movement of migrants between low- and middle- income countries29,30, so that a poor social response capacity within origin and destination countries is evidenced. This translates into less access to health, education and work; and increased susceptibility to irregular migration; migrant smuggling; and human trafficking31. Given this context and that Colombia is the main recipient of Venezuelan migrants, the migratory phenomenon can be seen and faced as an opportunity to progress at the regional level in the accomplishment of the SDGs, not only in those directly related to the health status of its inhabitants, but also in the tenth objective, which pertains to the reduction of inequalities. This would indicate that despite one of the main strategies of the National Development Plan 2018–2022 is to intervene in the determinants of inequality in the country32, migrants cannot be excluded from public policies in order to comply with the SDG targets, since the reduction of inequalities implies the social, economic and political inclusion of all people without any distinction; the guarantee of equality in opportunities; and the facilitation of an orderly, safe, regular and responsible migration and mobility of people.\n\nFinally, the present study shows the need for accurate, reliable and up-to-date information to facilitate the decision-making process and the design and assessment of public policies. This supports continuing to improve the already available information systems through more frequent updates and the inclusion of some basic demographic variables in the reports. Likewise, the design and implementation of better sources of information are required to comprehensively assess the health status of Venezuelan migrants and to overcome the obstacles of the currently available data. The latter could be achieved through a population survey focused on migrants, with the objective to assess their health status and other processes related to it, including migrants who meet inclusion criteria not related to their migratory status or to their history of previous contact with a health or justice entity. Such an information source would allow primary data to be obtained that is actually focused on assessing the health status of the Venezuelan migrant population, which would allow more valid estimates.\n\n\nStrengths and limitations\n\nWithin the strengths of this research are its originality and novelty, considering that literature on the Venezuelan migrants' health status is scarce and Colombia does not currently have an updated report of public health interest events that compares health indicators (especially those related to the SDGs) between Venezuelan migrants and the Colombian population. Therefore, our investigation contributes new information about this important topic, enabling a bigger and more updated picture of the migrants’ health status, which may enhance the restructuring of actions and the decision-making process to strengthen information systems and the responses from the health sector. In addition, the analysis was conducted with the latest data available in official records at the national level, which are one of the few sources of information at the moment, since other data are not official and no population surveys or other types of studies have been conducted to collect health status information about the Venezuelan migrant population. Finally, our results might help in assessing the degree of accomplishment of the SDGs in the Colombian territory and serve as guidance for future assessment of the health strategies used to comply with such goals.\n\nOn the other hand, there are also limitations that have to be discussed. First, some data from certain sources might be underestimated due to under-registration. This is the case for data from the INMLCF because they only register cases that have been denounced and whose victim did not desist from the medical-legal examination. Under-registration is also an issue in the estimations from Migración Colombia due to the highly-mobile migrant population (pendular and in-transit migrants) and to the high number or irregular migrants who use non-authorized crossing points in the borders. However, Migración Colombia tries to make the most accurate estimation possible of the number of Venezuelan migrants in Colombia by cross-referencing data from different information systems. Second, a selection bias is likely to affect the data used for the analysis, since the collected information comes from people who survived the migratory journey, while others die while trying to reach other countries21, and those who have had any contact with a health or justice institution. Therefore, subjects who cannot access health care or any other institution that makes a report are excluded from the official records. This is the case of irregular migrants, who can only access emergency health care services or who might not go to a health or justice facility out of fear of being deported. Additionally, the high mobility of the Venezuelan migrant population also hinders monitoring and data collection related to the health of these individuals. Third, an information bias (classification bias) cannot be ruled out in the estimations that were based on the information from vital statistics since “nationality” was not actually assessed; instead, the country of regular residence of the deceased person or of the mother of the deceased child/live birth was recorded. This does not allow the differentiation of Colombian returnees, foreigners who have been living in Colombia for several years or people who hold dual nationality (the latter hinders even more the distinction between Colombians and Venezuelan). This would be more relevant for the estimations of the Venezuelan population present in Colombia, inasmuch as their estimates are more unstable given their small denominators. Fourth, the total rates of alleged sexual felony, intimate partner violence and domestic violence could only be estimated since 2018 because that was the year from which the INMLCF started to register the victim’s nationality. Additionally, the rates by sex of the abovementioned indicators could only be estimated for 2018 because Migración Colombia did not provide the sex distribution of the total number of Venezuelan migrants in Colombia in the latest report of 2019. This is an aspect that should be enhanced for future publications.\n\n\nConclusion\n\nThe health indicators evaluated in this study show an apparent inequality in the fulfillment of some SDG targets between the Colombian population and the Venezuelan migrants in Colombia, with the former having better results in terms of lower maternal, neonatal, infant and under-five mortalities, and lower proportionate mortality due to undernourishment. In order to face the migratory phenomenon, the Colombian government has established a health response plan, which is still relatively recent, and for this reason the evaluation of the indicators analyzed here should be continued to make the relevant modifications and adjustments, since the absence of adjusted and correctly designed policies to respond to identified needs could increase the problem. On the other hand, although responding adequately and assertively to the Venezuelan migration is a complex situation, which involves an adaptation process spanning from the receiving population to its different institutions, it is important to bear in mind that the migrants' poor quality of life has an impact on the receiving population. Therefore, Colombia must remember that as long as a country generates more opportunities for incorporation into society and suitable environments and conditions, it will have greater possibilities of forming healthier population groups that minimize health costs in the medium and long term, and of having a higher compliance of the SDGs related to the health-disease process, thus improving the quality of life of its inhabitants.\n\n\nData availability\n\nAll data used to conduct this study are of public access and can be downloaded from the official websites of each institute without any formal request. The links to access them are shown below:\n\nVital statistics, DANE. “Defunciones” (deaths) or “No Fetal” (which refers to non-fetal deaths) and “Nacimientos” (live births) files from 2015–18: http://microdatos.dane.gov.co/index.php/catalog/MICRODATOS/about_collection/22/5.\n\nPopulation projections, DANE. “Serie de proyecciones de población por área y sexo” file: https://www.dane.gov.co/index.php/estadisticas-por-tema/demografia-y-poblacion/proyecciones-de-poblacion.\n\nFigures of injuries of external cause in Colombia, Violence Observatory (INMLCF). “Exámenes médico legales por presunto delito sexual. Colombia, 2018”; “Violencia intrafamiliar. Colombia, 2018” and “Información preliminar lesiones no fatales de causa externa en Colombia. Enero a diciembre de 2019” files: https://www.medicinalegal.gov.co/cifras-de-lesiones-de-causa-externa.\n\nInfographics of the number of Venezuelan migrants in Colombia (2018 and 2019; the latest 2019 infographic was released in early 2020), Migración Colombia: https://www.migracioncolombia.gov.co/index.php/infografias.", "appendix": "References\n\nMinisterio de Salud de la República de Colombia: Plan de Respuesta del Sector Salud al Fenómeno Migratorio. Bogotá, D.C. 2018; 49–69. Reference Source\n\nBanco Mundial: Migración desde Venezuela a Colombia: impactos y estrategia de respuesta en el corto y mediano plazo. 2018; 208. Reference Source\n\nMigración Colombia: Así ha sido la evolución de la crisis migratoria venezolana: corte 30 de septiembre de 2019. 2019; 9: 32. Reference Source\n\nMigración Colombia: Venezolanos en Colombia. 2019. Reference Source\n\nInstituto Nacional de Salud: Boletín N° 27 - Notificación eventos de interés en salud pública durante el fenómeno migratorio. 2020. Reference Source\n\nDepartamento Nacional de Planeación de la República de Colombia\|: Documento CONPES (Consejo Nacional de Política Económica y Social) 3950. Estrategia para la atención de la migración desde Venezuela. Bogotá, D.C. 2018. Reference Source\n\nOrganización Internacional para las Migraciones: La migración en la Agenda 2030. Ginebra, Suiza. 2018; 156. Reference Source\n\nGrupo Centro de Referencia Nacional sobre Violencia: Instituto Nacional de Medicina Legal y Ciencias Forenses Forensis - Datos para la vida. Bogotá, D.C. 2018. Reference Source\n\nMigración Colombia: Todo lo que debe saber sobre la migración Venezolana y no le han contado. 2018. Reference Source\n\nOrganización Panamericana de la Salud: Unidad de Información y Análisis de Salud. Glosario de Indicadores Básicos de la OPS. Iniciativa Regional de Datos Básicos en Salud; Glosario de Indicadores. Washington DC; 2015; 10–1. Reference Source\n\nOrganización Panamericana de la Salud: Nueva lista OPS 6/67 para la tabulación de datos de mortalidad CIE-10. Boletín Epidemiológico. 1999; 20(3): 4–9. Reference Source\n\nMinisterio de Salud de la República de Colombia: Resolución número 8430 de 1993. Colombia; 1993; 1–19. Reference Source\n\nAngarita Álvarez OL: Bioética en estudios de vida real retrospectivos con fuente secundaria. Universidad de La Sabana. Universidad de La Sabana; 2019. Reference Source\n\nDoocy S, Page KR, de la Hoz F, et al.: Venezuelan Migration and the Border Health Crisis in Colombia and Brazil. J Migr Hum Secur. 2019; 7(3): 79–91. Publisher Full Text\n\nProfamilia: Evaluation of unmet sexual and reproductive health needs of the Venezuelan migrant population in four cities of the Colombia-Venezuela border: Arauca, Cúcuta, Riohacha, and Valledupar. Bogotá, D.C.; 2019; 34–36. Reference Source\n\nGarcía J, Correa G, Rousset B: Trends in infant mortality in Venezuela between 1985 and 2016: a systematic analysis of demographic data. Lancet Glob Heal. 2019; 7(3): e331–e336. PubMed Abstract \| Publisher Full Text\n\nProyecto Migración Venezuela: Necesidades en salud de la población migrante venezolana en Colombia. 2019. Reference Source\n\nAndersen RM: Revisiting the behavioral model and access to medical care: does it matter? J Health Soc Behav. 1995; 36(1): 1–10. PubMed Abstract\n\nJaime RT, Julio SC: Venezuela’s migration crisis: a growing health threat to the region requiring immediate attention. J Travel Med. 2018; 26(2): tay141. PubMed Abstract \| Publisher Full Text\n\nPage KR, Doocy S, Reyna Ganteaume F, et al.: Venezuela’s public health crisis: a regional emergency. Lancet. 2019; 393(10177): 1254–60. PubMed Abstract \| Publisher Full Text\n\nHuman Rights Watch: El éxodo venezolano - Urge una respuesta regional ante una crisis migratoria sin precedentes. 2018. Reference Source\n\nMinisterio de Salud y Protección Social: Circular externa 012. Colombia; 2017. Reference Source\n\nMinisterio de Salud y Protección Social: Circular externa 029. Colombia; 2017. Reference Source\n\nCala-Carrillo MJ, García-Jiménez M, González-Monteagudo J, et al.: Migrant women’s experiences of sexual and gender-based violence and help-seeking journeys: Focus on. Seville, Spain;. Publisher Full Text\n\nMiranda M: Gender-Based Violence: A Global Crisis that is Handled Ineffectually. Trinity College; 2020. Reference Source\n\nZulver J, Idler A: Gendering the border effect: the double impact of Colombian insecurity and the Venezuelan refugee crisis. Third World Q. 2020; 1–19. Publisher Full Text\n\nAsociación Profamilia y Oficina de los Estados Unidos de Asistencia para Desastres en el Extranjero (OFDA-USAID): Desigualdades en salud de la población migrante y refugiada venezolana en Colombia ¿Cómo manejar la respuesta local dentro de la emergencia humanitaria? Bogotá, D.C.; 2020. Reference Source\n\nOrganización Panamericana de la Salud, Organización Mundial de la Salud: Estrategia para el acceso universal a la salud y cobertura universal de salud. 53° Consejo Directivo de la OPS 66a Sesión del Comité Regional de la OMS para las Américas. Washington, D.C.; 2014. Reference Source\n\nStefoni C: Panorama de la migración internacional en América del Sur. Documento elaborado en el marco de la Reunión Regional Latinoamericana y Caribeña de Expertas y Expertos en Migración Internacional preparatoria del Pacto Mundial para una Migración Segura, Ordena. Población y Desarollo. 2018; 123. Reference Source\n\nFernández Castilla R: Migraciones y Remesas en el Contexto de la Globalización. Okinawa; 2005. Reference Source\n\nInternational Organization for Migrations (IOM): La Migración Sur-Sur: Asociarse de Manera Estratégica en pos del desarrollo [Internet]. 2014. Reference Source\n\nDepartamento Nacional de Planeación de la República de Colombia: Bases del Plan Nacional de Desarrollo 2018-2022: Pacto por Colombia, pacto por la equidad. Bogotá, D.C.; 2019. Reference Source" }	[ { "id": "83119", "date": "01 Jun 2021", "name": "Theodoros Fouskas", "expertise": [ "Reviewer Expertise Migrants", "Health" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript focuses on the comparison of some health indicators related to the Sustainable Development Goals between the Venezuelan migrant population and the Colombian population. This is a very pertinent issue and the article deals well with this.\n\nIt offers a robust theoretical frame and a well-organized and convincing discussion of the findings.\n\nThe manuscript is informative and its reading enjoyable. The author(s) has/have paid attention to the clarity of expression and readability, such as sentence structure. The quality of communication is good.\n\nThe theoretical part is well developed. A clear stating and focusing of the argument is provided.\n\nThe author(s) addresses a significant research subject and presents interesting material. This article could be a starting point for further research.\n\nMethods are appropriate and the fit between theoretical discussion and methodology is well formulated.\n\nThe manuscript highlights the central literature concerning the theme.\n\nThe conclusions are linked to the hypothesis and background characteristics incorporated into the results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-684
https://f1000research.com/articles/9-610/v1	15 Jun 20	{ "type": "Case Report", "title": "Case Report: Dilated cardiomyopathy with biventricular thrombus secondary to impaired coagulation in a patient with HIV", "authors": [ "Chetan Brahma Kammari", "Suhasini Rallabandi", "Harsha Rallabandi", "Subba Rao Daggubati", "Sreedhar Adapa", "Srikanth Naramala", "Venu Madhav Konala", "Suhasini Rallabandi", "Harsha Rallabandi", "Subba Rao Daggubati", "Sreedhar Adapa", "Srikanth Naramala", "Venu Madhav Konala" ], "abstract": "Human immunodeficiency virus (HIV) infection is a known hypercoagulable state with venous thromboembolism with a high mortality rate compared to the general population. The homeostatic balance in HIV infected patients improves with treatment compared to those who are not. A decreased hypercoagulable state noted by low levels of Von Willebrand factor, factor VIII and d-dimer levels along with higher protein C and S activity in patients on treatment suggests that hypercoagulable state is partially correctable with highly active antiretroviral therapy. HIV with heart muscle involvement can present as myocarditis or as dilated cardiomyopathy with left or right ventricular dysfunction. Here we present a case of a 57-year-old man with a known history of HIV infection, noncompliant with medical therapy presenting with dilated cardiomyopathy with biventricular thrombi with reduced protein C, protein S, and Antithrombin III levels.", "keywords": [ "HIV", "Hypercoagulable", "Ventricular", "thrombus", "protein c", "protein s", "antithrombin 3" ], "content": "Introduction\n\nHuman immunodeficiency virus (HIV) infection is a well-known hypercoagulable state associated with venous thromboembolism with high mortality risk compared to the general population1,2. HIV with heart muscle involvement can present as myocarditis or as dilated cardiomyopathy with left or right ventricular dysfunction3. Here we present a case of a patient infected with HIV presenting with dilated cardiomyopathy with biventricular thrombi secondary to reduced protein C, protein S, and antithrombin III levels. On review of the literature, we were able to find only one similar presentation where a patient with HIV has cardiomyopathy with biventricular thrombosis4.\n\n\nCase report\n\nThe patient is a 57-year-old Caucasian male with a known past medical history of human immunodeficiency virus (HIV) noncompliant with medical therapy and hyperlipidemia, who presented to the emergency department with shortness of breath, hypoxia with oxygen saturation of 70%, pleuritic chest pain and a syncopal episode with fall. The patient denied any significant family, surgical, or social history. He was treated for pneumonia six weeks prior to presentation with ceftriaxone 1 gram daily and azithromycin 500mg daily for 5 days, and since then he has been experiencing exertional dyspnea. The patient denied orthopnea or paroxysmal nocturnal dyspnea. He had a syncopal episode at home with fall and hitting the left side of the chest on a barstool causing pleuritic chest pain. Chest pain was non-exertional. The patient's level of activity has significantly diminished over the last six weeks, and he was unable to perform his daily activities without difficulty. The patient admitted that he had previous episodes of syncope that occur with little or no warning signs except for mild dizziness before passing out. He denied any associated chest tightness, palpitations, headache, and stool or urine incontinence. The physical examination was significant for chest wall tenderness with a normal cardiorespiratory exam.\n\nLaboratory findings showed mildly elevated troponin. An echocardiogram demonstrated biventricular dilatation with ejection fraction (EF) of 30% and compelling evidence for the presence of thrombus in the apex of both ventricles and free wall of the right ventricle (as shown in Figure 1–Figure 4). Echocardiogram did not demonstrate any spontaneous echo contrast, suggesting severely diminished ejection fraction and stagnation of blood flow. Orthostatic vitals were normal, and the patient did not experience any arrhythmias on telemetry ruling them out as a cause for syncope. Syncope was later presumed to be likely secondary to a low flow state from reduced EF. The patient denied any prior history of deep vein thrombosis, transient ischemic attack, or stroke. CT chest with contrast did not show any evidence of pulmonary embolism but showed diffuse cardiomegaly (Figure 5 and Figure 6). Given the presence of biventricular thrombus, the patient was evaluated for the hypercoagulable state. Results showed low Protein C, protein S, and antithrombin III levels. Factor V Leiden and lupus anticoagulant were normal. The laboratory findings are summarized in Table 1.\n\nCardiology initially considered cardiac catheterization to delineate the patient’s coronary anatomy for the potential need for revascularization, but instead decided to perform stress myocardial perfusion study to prevent thromboembolic events and strokes that can be associated with the procedure. The nuclear stress test was negative for reversible ischemia. There was no evidence of fixed defects on the stress test, suggesting no evidence of prior myocardial infarction or scar tissue within the myocardium. The patient was started on Lasix 20mg oral daily, metoprolol succinate 25mg oral daily, and low-dose Lisinopril 2.5mg oral daily. Novel anticoagulants (NOACs) are not recommended for mural thrombus; therefore, the patient was started on a therapeutic dose of low molecular weight heparin 60mcg subcutaneous twice daily and bridged with warfarin 5mg oral daily. Heparin was discontinued once his International Normalized Ratio (INR) was therapeutic. One week after the admission, the patient was ready to be discharged. His symptoms had significantly improved with no further syncopal episodes or exertional dyspnea. His ambulatory oxygen saturations were normal on room air. The patient was advised to follow up with infectious diseases to initiate highly active antiretroviral therapy (HAART) therapy for his HIV, cardiology and hematology for continued care.\n\n\nDiscussion\n\nHIV infection is a well-known hypercoagulable state with a frequency of thrombotic events recognized in the range of 0.19% to 7.63% per year1. Compared to the general population of the same age, the risk of arterial and venous thrombosis in HIV infected patients is increased two to tenfold. One possible explanation could be due to the presence of multiple comorbidities and baseline increased inflammatory/hypercoagulable state. Risk factors like low CD4 cell count, especially in the presence of clinical acute immunodeficiency syndrome (AIDS), protein S deficiency, and protein C deficiency, have demonstrated the strongest association with venous thromboembolism. Other less significant and controversial risk factors include protease inhibitor therapy, opportunistic infections, and positive antiphospholipid antibodies, including anticardiolipin antibodies and lupus anticoagulant1,5. Thrombophilic abnormalities in total platelet count, protein C, and S activity are directly correlated to CD4 count6, and their frequency increases as the patient progresses to AIDS7. HIV infection with venous thromboembolism has a high mortality rate compared to the general population2.\n\nThe homeostatic balance in HIV-infected patients varies if they are on HAART therapy vs. not being on treatment. The indicators of hypercoagulable state like the activity of the Von Willebrand factor, levels of Factor VIII, and D dimer are lower in patients on treatment. Also, anticoagulant proteins like protein C and S activity are higher in patients on therapy, suggesting the hypercoagulable state is partially correctable with HAART. Although patients on HAART therapy have a lower prevalence of coagulation abnormalities, many show the persistent procoagulant state as evident by increased endothelial cell activation and high APCsr when compared to the general population. Continued coagulation markers abnormalities have been observed in HIV-infected individuals before and after the initiation of HAART8. Even patients who are newly started on HAART therapy showed marked improvement in the coagulation profile but still different from the general population, indicating a persistent abnormal homeostatic balance9. In addition, it has been previously observed that most HIV positive patients with low protein S levels also had mutations in exon 15 of PROS 1 gene warranting further investigation10.\n\nCardiac dysfunction in HIV-infected patients evidenced by congestive heart failure is a well-documented finding. Even in asymptomatic HIV patients, an echocardiographic and echo-doppler examination has shown evidence of early signs of impaired systolic and diastolic function, suggesting an early involvement of the heart in HIV disease11. Cardiomyopathy associated with HIV may be related to the autoimmune process induced by HIV in conjugation with other cardio tropic virus or could be direct action on the heart muscle12. It is shown that the incidence of dilated cardiomyopathy in HIV is 17.6%13. HIV with heart muscle involvement can present as myocarditis or dilated cardiomyopathy with left or right ventricular dysfunction, the pathogenesis of which seems to relate to the coinfection with other infectious agents3. Isolated right and left heart dysfunction have no direct correlation to the CD4 count and does not carry adverse prognostic implications14.\n\nLong term anticoagulation may be beneficial in HIV infected patients to prevent future thromboembolic events as most of the contributing risk factors are often irreversible. Since there is a possibility of interactions between warfarin and antiretroviral therapy, health care providers should be watchful of consequent dangerously high or low INRs when giving warfarin to patients undergoing antiretroviral therapy1. It is shown that newer anticoagulants can be used with antiretroviral therapy without any noticeable interactions15.\n\n\nConclusion\n\nIn our patient, a bi-ventricular thrombus is likely the result of the hypercoagulable state along with severe ventricular dysfunction with dilated cardiomyopathy due to underlying HIV infection. Systemic or pulmonary embolization was not seen in our patient, as reported in the past as an associated finding16. Physicians caring for patients with HIV should always consider thrombotic and thromboembolic events in the differential besides known malignancies and opportunistic infections when treating patients with unexplained dyspnea or hypoxia, especially in young males.\n\n\nConsent\n\nWritten informed consent for the publication of the case report and any associated images was obtained from the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nBibas M, Biava G, Antinori A: HIV-associated venous thromboembolism. Mediterr J Hematol Infect Dis. 2011; 3(1): e2011030. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFultz SL, McGinnis KA, Skanderson M, et al.: Association of venous thromboembolism with human immunodeficiency virus and mortality in veterans. The American journal of medicine. 2004; 116(6): 420–3. PubMed Abstract \| Publisher Full Text\n\nKhunnawat C, Mukerji S, Havlichek D Jr, et al.: Cardiovascular manifestations in human immunodeficiency virus-infected patients. Am J Cardiol. 2008; 102(5): 635–42. PubMed Abstract \| Publisher Full Text\n\nNkoke C, Kuate LM, Luchuo EB, et al.: Biventricular thrombi in dilated cardiomyopathy in a patient with human immunodeficiency virus infection: a case report. BMC Res Notes. 2015; 8(1): 168. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKiser KL, Badowski ME: Risk factors for venous thromboembolism in patients with human immunodeficiency virus infection. Pharmacotherapy. 2010; 30(12): 1292–302. PubMed Abstract \| Publisher Full Text\n\nKhare S, Kushwaha R, Kumar A, et al.: Prothrombotic state in HIV: A study on protein C, protein S, homocysteine and correlation with CD4 counts. Indian J Med Microbiol. 2018; 36(2): 201–206. PubMed Abstract \| Publisher Full Text\n\nLijfering WM, Sprenger HG, Georg RR, et al.: Relationship between progression to AIDS and thrombophilic abnormalities in HIV infection. Clinical Chemistry. 2008; 54(7): 1226–33. PubMed Abstract \| Publisher Full Text\n\nJong E, Louw S, Meijers JC, et al.: The hemostatic balance in HIV-infected patients with and without antiretroviral therapy: partial restoration with antiretroviral therapy. AIDS Patient Care STDS. 2009; 23(12): 1001–7. PubMed Abstract \| Publisher Full Text\n\nJong E, Louw S, Gorp ECM: The effect of initiating combined antiretroviral therapy on endothelial cell activation and coagulation markers in South African HIV-infected individuals. Thromb Haemost. 2010; 104(6): 1228–34. PubMed Abstract \| Publisher Full Text\n\nDi Stefano M, D’Andrea G, Zoboli F, et al.: Preliminary Data From the Study of Coagulative Profile of HIV Infected Individuals Suggest a Role For Point Mutations in the Gene in Protein S Deficiency in Individuals Undergoing Highly Antiretroviral Therapy. Open AIDS J. 2018; 12: 6–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarbaro G, Barbarini G, Di Lorenzo G: Early impairment of systolic and diastolic function in asymptomatic HIV-positive patients: a multicenter echocardiographic and echo-doppler study. AIDS Res Hum Retroviruses. 1996; 12(16): 1559–63. PubMed Abstract \| Publisher Full Text\n\nBarbaro G, Di Lorenzo G, Grisorio B, et al.: Cardiac involvement in the acquired immunodeficiency syndrome: a multicenter clinical-pathological study. AIDS Res Hum Retroviruses. 1998; 14(12): 1071–7. PubMed Abstract \| Publisher Full Text\n\nJain N, Reddy DH, Verma SP, et al.: Cardiac abnormalities in HIV-positive patients: results from an observational study in India. J Int Assoc Provid AIDS Care. 2014; 13(1): 40–6. PubMed Abstract \| Publisher Full Text\n\nCurrie PF, Jacob AJ, Foreman AR, et al.: Heart muscle disease related to HIV infection: prognostic implications. BMJ. 1994; 309(6969): 1605–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNatarajan P, Joolhar F, Thangarasu S, et al.: Embolizing Massive Right Atrial Thrombus in a HIV-Infected Patient. J Investig Med High Impact Case Rep. 2018; 6: 2324709618802871. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMissault L, Koch A, Colardyn F, et al.: Biventricular thrombi in dilated cardiomyopathy: massive simultaneous pulmonary and systemic embolisation. Eur Heart J. 1994; 15(5): 713–4. PubMed Abstract \| Publisher Full Text" }	[ { "id": "64868", "date": "29 Jun 2020", "name": "Raju Vaddepally", "expertise": [ "Reviewer Expertise Hematology", "Medical Oncology Thoracic and head and neck medical Oncology", "Immunotherapy", "Targeted therapy", "Precision Medicine." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very nice case review on alterations in homeostatic mechanism in HIV. I tend to agree that there are inflammatory changes in HIV patients that deranges the the coagulation equilbrium and there are limitations in available literature on what exactly drives the prothrombotic features in such patients.\n\nHaving said that in this case, the patient seems to have dilated cardiomyopathy which largely seems to have driven the thrombosis and the case has been very well elaborated with description, pictures, and lab assay. However, the low protein C, S, and Antithrombin evidenced here rather depicts the state of thrombus consuming the factors. It would have been nice if they had follow up lab assay follow on these protein C, S, and Antithrombin and if the numbers continue to remain low after 3-6 months from the time of thrombosis then one could make a strong argument that the coagulation factor deficiencies could have accentuanted the thrombosis but it is unclear at this time based on the available labs.\nThe authors should be commended for ensuring the APAS etiology has been ruled out which would be an important etiology in HIV patients. However, I do suggest trimming the case report content especially in the first paragraph where there is redundancy in non-pertinent information. Overall, I feel this contributes some knowledge to existing data of HIV and hypercoagulability.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "5676", "date": "07 Jul 2020", "name": "Chetan Kammari", "role": "Author Response", "response": "I agree with the reviewer's comment that low protein C, low protein S, and anti-thrombin deficiency could represent the state of thrombus consuming the factors. It is not 6 months yet and the patient continues to be followed up as an outpatient. Modified in the manuscript. Modified in the manuscript as below - We think that low protein C, low protein S levels, and antithrombin-III deficiency could contribute to thrombus formation if truly positive in addition to the risk factors discussed above. However, it could represent a thrombus consuming the factors, and they will be repeated six months to confirm it. We trimmed the case report content in the first paragraph as advised. First Paragraph - The patient is a 57-year-old Caucasian male with a known past medical history of the human immunodeficiency virus (HIV) non-compliant with medical therapy and hyperlipidemia, who presented to the emergency department with shortness of breath, hypoxia with oxygen saturation of 70%, pleuritic chest pain and a syncopal episode with fall. The patient denied any significant family, surgical, or social history. He was treated for pneumonia six weeks before presentation with antibiotics, and since then, he has been experiencing exertional dyspnea. Patient unable to do his activities of daily living due to exertional dyspnea. The patient denied orthopnea or paroxysmal nocturnal dyspnea. He had a syncopal episode at home with fall resulting in left pleuritic chest pain. The patient admitted that he had previous syncope episodes that occur with little or no warning signs except for mild dizziness before passing out. The physical examination was significant for chest wall tenderness with a normal cardiorespiratory exam." } ] }, { "id": "64869", "date": "01 Jul 2020", "name": "Satyanarayana Vaidya", "expertise": [ "Reviewer Expertise Nephrology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAuthors have done a commendable job in writing a detailed case report depicting Increased thrombogenicity and a low blood flow state in HIV a infected patient as primary mechanisms of the virchow triad causing biventricular thrombosis. The tables, figures provide great deal of detailed information regarding the location of thrombus.\nI would like to highlight certain important points and would encourage the inclusion of those in the case report\nThe low flow state due to cardiomyopathy seems to have been a primary mechanism here causing biventricular thrombosis. If patient had a transesophageal echocardiography to exclude a patent foramen ovale which can result in paradoxical embolism or deep vein assessment with a venous doppler, inclusion of that information would be critical to exclude any possibility of venous thromboembolism\n\nPatient seems to have a decent CD4 count of 815 cells/microliter despite being non-compliant with Antiretroviral therapy. Inclusion of HIV viral load would give more information as there is evidence regarding high viral loads and low CD4 counts being related to increased thrombogenicity. In addition, presence of a reasonable CD4 cell count argues against the possibility of HIV associated nephropathy which can cause significant proteinuria causing loss of anticoagulant proteins causing a thrombogenic state. Information regarding protein: creatinine ratio would have evaluated that possibility.\n\nOne would always make an argument of low protein C, protein S levels being low in the setting of acute thrombosis. Following them after a period of at least 6 months would reveal the true levels and differentiate between true deficiency vs due to consumption due to thrombosis.\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? Partly", "responses": [ { "c_id": "5677", "date": "07 Jul 2020", "name": "Chetan Kammari", "role": "Author Response", "response": "We agree with the reviewer's comment that the low flow state due to cardiomyopathy could have contributed, we think it is multifactorial in this patient with low flow state as well as possible protein deficiencies if truly positive contributing to it. Unfortunately, a TEE was not done during that hospitalization. I agree with the reviewer's comment that low protein C, low protein S, and anti-thrombin deficiency could represent the state of thrombus consuming the factors. It is not 6 months yet and the patient continues to be followed up as an outpatient. Modified in the manuscript. We thank the reviewer for pointing out normal CD4 count - low CD4 count is one of the risk factors, but there additional risk factors in HIV infected patients like a protein C deficiency, protein S deficiency, antithrombin deficiency, antiphospholipid antibody syndrome, endothelial dysfunction in addition to the traditional risk factors such as obesity, cigarette smoking, hypertension, immobilization. We think that thrombus is multifactorial in this patient. We did not think the patient has HIV-induced nephropathy resulting in nephrotic syndrome - hence it was not investigated further, no protein creatinine ratio was done during the admission." } ] } ]	1	https://f1000research.com/articles/9-610
https://f1000research.com/articles/9-682/v1	07 Jul 20	{ "type": "Brief Report", "title": "Reducing childhood obesity: evaluation of an Early Years Nutrition programme in a deprived London borough", "authors": [ "Priyanka Patil", "Emma C. Alexander", "Meghan Cupp", "Monica Lakhanpaul", "Meradin Peachey", "Alexander Light", "Logan Manikam", "Priyanka Patil", "Emma C. Alexander", "Meghan Cupp", "Monica Lakhanpaul", "Meradin Peachey", "Alexander Light" ], "abstract": "Background: Childhood obesity is a growing global health concern, with far-reaching implications on health in childhood and in later life. Early intervention strategies are key to reducing childhood obesity. This study aims to evaluate the implementation of an Early Years Nutrition programme in the London Borough of Newham’s children’s centres. Methods: A service evaluation of the Early Years Nutrition programme was conducted at children’s centres within the borough. Information was collected on the sessions provided to parents by staff, breastfeeding promotion and nutritional topics the centres were displaying. Nutritional activities in each centre were assessed for compliance with the National Institute for Health and Care Excellence (NICE) guidelines. Results: Eight out of eleven (72.7%) centres participated. Parent sessions focused mostly on oral health (n=4/8, 50.0%). Display board topics most commonly related to generic health and wellbeing (n=4/8, 50.0%). All centres displayed the UNICEF breastfeeding logo and complied with the NICE guidelines for nutritional activities. Conclusions: The programme is consistent with NICE guidelines in the centres evaluated; however, further acquisition of data on obesity-associated factors specific to communities and preventive measures for reducing childhood obesity, such as increased parental and community engagement, promotion of breastfeeding and improved staff training, will help tailor similar programmes elsewhere with higher social and cultural acceptance.", "keywords": [ "childhood obesity", "nutrition", "children", "London", "evaluation" ], "content": "Introduction\n\nObesity in childhood has a significant impact on an individual’s lifelong health and well-being. The associated risks of excess weight in childhood include an increased risk of becoming overweight as an adult, and of developing serious health conditions1. Although childhood obesity is considered as one of the most serious challenges of the 21st century, policy and research efforts have not been successful in halting the rise of overweight and obesity, resulting in almost a 20% rise in obese and overweight children in the UK over the past two decades2.\n\nThe UK government introduced its childhood obesity strategy ‘A Plan for Action’, in 20163. This plan acknowledged that ‘Long-term, sustainable change will only be achieved through early intervention and active engagement of communities, families, schools and individuals.’ One such effort is the Early Years Nutrition programme, a local strategy in a London borough (Newham). Newham has an ethnically diverse population and the third highest rate of childhood obesity amongst all London boroughs, which is increasing steadily4. In light of this, Newham’s Childhood Obesity Action Plan was introduced in 2017 to define the borough’s vision for addressing childhood obesity5. The current study aimed to assess the implementation of this programme through an evaluation of services offered in Newham's children's centres and provide recommendations for wider implementation of similar programmes.\n\n\nMethods\n\nAll Newham's children's centres were invited to participate in this study (n=11). Our objectives were to conduct a service evaluation of what information about healthy eating (topics) is provided on display boards in early years settings against set standards by National Institute for Health and Care Excellence (NICE) guidelines. In addition, we aimed to determine what sessions the centres offer in their timetable for parents and whether centres are displaying the UNICEF’s ‘you are welcome to breastfeed here’ logo to promote breastfeeding6.\n\nData collection occurred from January 1, to March 31, 2018. Information about healthy eating topics was collected through photographs and field notes of the display boards of each of the centres, while information about parent sessions was collected from the centre staff and available information pamphlets at the centres.\n\nAdditionally, we assessed whether nutrition activities (both parent sessions and nutritional topics) of each centre complied with the maternal and child nutrition guidelines (PH11) from the NICE7. These guidelines set out the key recommendations for proper nutrition during pregnancy and for children under five years of age and highlight interventions aimed at optimising nutrition for children. The initiatives within Newham’s early years nutrition programme were identified by the research team and mapped/classified against the 22 NICE recommendations for maternal and child nutrition to determine where Newham’s offerings were compliant and to identify areas for improvement.\n\nSince this study was a routine service evaluation, we used the Health Research Authority decision tool to confirm that Research Ethical Committee approval was not required8.\n\n\nResults\n\nOut of the eleven centres in Newham, eight (72.7%) centres participated (Table 1). The three remaining centres failed to respond when approached to participate. Of the remainder, one was newly opened and therefore did not have display boards. Findings from assessing the display boards revealed that the most frequently displayed topics were related to generic health and wellbeing (4 centres, 50.0%), and breastfeeding (3, 37.5%) (Table 1).\n\nCentres were assessed to see whether they displayed the UNICEF breastfeeding logo, what sessions and workshops were available to parents, and nutritional display board topics from January to March 2018.\n\nOn assessing the parent sessions offered, we observed that they were varied, with no single session topic widely offered across centres (Table 1). The most frequent sessions offered were oral health (4, 50.0%) and nutrition (3, 37.5%) workshops. Three centres (37.5%) did not offer any relevant sessions during this period.\n\nAll centres displayed the UNICEF breastfeeding logo.\n\nAssessment of the centres’ display information revealed that the Early Year Nutrition programme is meeting the NICE recommendations for child nutrition, achieving all recommendations within the scope of this study (Table 2).\n\n\nDiscussion\n\nThe display boards were designed to encourage parents to visit the centre and join discussions about health topics displayed. Existing research has demonstrated that training staff from children’s centre in delivering key evidence-based healthy eating and physical activity messages results in an increase in adoption of formal nutrition and physical activity policies within the early years setting9. It is also evident that, in addition to the adoption of such policies, staff training has resulted in key benefits for children, such as improved nutrition and increased energy levels10.\n\nIn general, we recommend that the children’s centres should aim to offer more workshops, ideally a schedule with topics standardised across all boroughs to ensure consistency in the information delivered to parents. These sessions should also cater to specific age groups in children. Programmes targeted at families with children under age five that aim to promote healthy choices, have demonstrated positive outcomes in terms of improved health behaviours11. Furthermore, a previous study suggests that home-based interventions delivered to target families can reduce the Body Mass Index (BMI) status for children under the age of two12. Therefore, programmes aimed at increasing health visitor capacity to intervene around nutrition and obesity issues through increased parental engagement may be a potential method of reducing later childhood obesity in the UK. However, there is little longitudinal evidence about the role of the UK’s health visitors in reducing childhood obesity at older ages, and thus further research into their significance in preventing and reversing childhood obesity is required.\n\nAlthough conducting parental interviews was not within the scope of our study, we recommend developing nutritional programmes that focus on building a bridge between community centres or service providers and parents, with a goal of improving communication about child nutrition and promotion of child health. This should include regular engagements with parents and young individuals in the community to educate them and gather essential feedback regarding the services and knowledge offered. Previous studies have proved the importance of service user feedbacks as these are pivotal in enforcing changes and improving the delivery of programmes13,14. A 2010 systematic review also concluded that parental engagement resulted in increased health-related behavioural change, with better outcomes associated with increased parental engagement compared to controls15. Furthermore, we recommend programme planners take a ‘participatory learning approach’ to intervention design, as there is evidence suggesting its benefits in communities for the success of such interventions16. This approach involves members from the community and increases the chances of adherence to promoted advice, as they are specifically developed with the community in question, thus making them socially and culturally acceptable16.\n\nDisplaying the UNICEF breastfeeding logo is a simple intervention to support UNICEF’s Baby Friendly Initiative and promote breastfeeding both in the children’s centre and in general by encouraging mothers and families to pursue this as a feeding choice. Since the Initiative was established, breastfeeding initiation rates have increased by 20%17 and there is evidence suggesting a correlation between breastfeeding and decreased risk of childhood obesity18. This is especially important as the UK has a relatively low breastfeeding rate, which is amongst the worst in Europe19. Currently there aren’t any studies exploring the strength of displaying breastfeeding logos, but given the benefits of breastfeeding, further research is warranted that looks at the impact of local supportive initiatives, and particularly if displaying such logos in public places can actually influence breastfeeding behaviour on a local level.\n\nOverall, the Early Years Nutrition programme is meeting the NICE recommendations for child nutrition, with efforts to address all recommendations within the scope of this study (Table 2). These evidence-based guidelines highlight the key recommendations for optimal nutrition for children7 and should form the basis for the benchmarking of activities to monitor what services are available within the boroughs and assess if they meet NICE guidance.\n\nDue to no response, some children’s centres were excluded from the study. It is possible that non-responders may have had poor adherence to guidelines and hence did not reply to our request. Private and voluntary settings providing similar services were not included. In addition, it was not possible to assess the nutritional quality of the food provided in the children’s centres. Furthermore, parental feedback on the Early Years Nutrition programme was not collected.\n\n\nConclusion\n\nThis evaluation indicates that the Early Years Nutrition programme is consistent with NICE guidelines, addressing all 18 relevant recommendations. However, there are several important key areas for development for programme planners delivering such services. These include improved staff training to ensure accuracy and standardisation of promoted information, consistency in sessions delivered to parents, not just within the borough, but within all similar services across the country and further exploring the role of health visitors in promoting nutritional and lifestyle changes to reduce childhood obesity. In addition, we suggest further engagement with parents and young people regarding the services offered, and the potential for applying the participatory approach to intervention design for the success of such programmes in communities. This may further strengthen delivery of the programmes, and contribute to their efficacy and further implementation of similar childhood obesity programmes in other settings.\n\n1. Childhood obesity is rising;\n\n2. Effective early interventions are key to prevent the rise of childhood obesity;\n\n3. Targeting parental perceptions with appropriate knowledge around early nutrition acknowledging their cultural beliefs and practices is required;\n\n4. Routine service evaluations of community health programmes are essential to improve programme delivery;\n\n5. Improved staff training and increased community engagement is key for a successful programme\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Acknowledgements\n\nThe authors would like to thank Newham Council and all participating children’s centres. Authors would also like to thank Yancy Jensen (Newham Council & UCL) and Roeann Osman (UCL) for their contributions to the manuscript.\n\n\nAuthor contributions\n\nAll authors were involved in, at various stages, conception of the work and modification to the design. Individual lead responsibilities are listed in the Author roles section. All authors additionally contributed to revisions and the final draft, and gave approval of the version to be published and agree to be accountable for all aspects of the work.\n\n\nReferences\n\nDietz WH: Health consequences of obesity in youth: childhood predictors of adult disease. Pediatrics. 1998; 101(3 Pt 2): 518–525. PubMed Abstract\n\nStatistics on obesity, physical activity and diet. England, 2019. Reference Source\n\nDepartment of Health: Prevalence of Childhood Obesity. Borough, Ward and MSOA. 2018. Accessed 14 Jun 2018. Reference Source\n\nTrust for London: Child obesity. 2020. Reference Source\n\nNewham London: Children and Young People’s Plan 2015-2018. Newham London. 2015. Reference Source\n\nUNICEF: Welcome to breastfeed here posters. Accessed 14 Jun 2018. Reference Source\n\nNational Institute for Health and Care Excellence: Maternal and child nutrition Public health guideline [PH11]. 2014. Accessed 14 Jun 2018. Reference Source\n\nNHS Health Research Authority: Is my study research? Accessed 5 Jul 2018. Reference Source\n\nTomayko EJ, Prince RJ, Hoiting J, et al.: Evaluation of a multi-year policy-focused intervention to increase physical activity and related behaviors in lower-resourced early care and education settings: Active Early 2.0. Prev Med Rep. 2017; 8: 93–100. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWard DS, Welker E, Choate A, et al.: Strength of obesity prevention interventions in early care and education settings: a systematic review. Prev Med. 2017; 95(suppl): S37–S52. PubMed Abstract \| Publisher Full Text\n\nWillis TA, George J, Hunt C, et al.: Combating child obesity: impact of HENRY on parenting and family lifestyle. Pediatr Obes. 2014; 9(5): 339–350. PubMed Abstract \| Publisher Full Text\n\nWen LM, Baur LA, Simpson JM, et al.: Effectiveness of home based early intervention on children’s BMI at age 2: randomised controlled trial. BMJ. 2012; 344: e3732. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDev D, Byrd-Williams C, Ramsay S, et al.: Engaging Parents to Promote Children’s Nutrition and Health. Am J Health Promot. 2017; 31(2): 153–162. PubMed Abstract \| Publisher Full Text\n\nKruk JJ, Kortekaas F, Lucas C, et al.: Obesity: A systematic review on parental involvement in long-term European childhood weight control interventions with a nutritional focus. Obes Rev. 2013; 14(9): 745–760. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHingle MD, O’Connor TM, Dave JM, et al.: Parental involvement in interventions to improve child dietary intake: a systematic review. Prev Med. 2010; 51(2): 103–111. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMartin J, Fottrell E, Black G, et al.: Adaptations to the participatory learning and action cycle in resource-limited settings: an observational study. Lancet. 2017; 390(Supplement 3): S63. Publisher Full Text\n\nUNICEF: What is baby friendly? 2018. Accessed 14 Jun 2018. Reference Source\n\nRenfrew MJ, Spiby H, D’Souza L, et al.: Rethinking research in breast-feeding: a critique of the evidence base identified in a systematic review of interventions to promote and support breast-feeding. Public Health Nutr. 2017; 10(7): 726–732. PubMed Abstract \| Publisher Full Text\n\nCattaneo A, Burmaz T, Arendt M, et al.: Protection, promotion and support of breast-feeding in Europe: progress from 2002 to 2007. Public Health Nutr. 2010; 13(6): 751–759. PubMed Abstract \| Publisher Full Text" }	[ { "id": "86254", "date": "15 Jun 2021", "name": "Oliver J Canfell", "expertise": [ "Reviewer Expertise Obesity", "childhood obesity", "nutrition", "dietetics" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary This brief report from Patil and co-authors describes a service evaluation of an Early Years Nutrition Programme implemented in eleven children’s centres in Newham – an underserved London borough. The evaluation involved assessing compliance between the implemented Early Years Nutrition Programme and the National Institute for Health and Care Excellence (NICE) guidelines. Patil et al. reported the programme is consistent with the NICE guidelines.\nOverall, the study purpose is clear – appropriate for a brief report. I have major and minor concerns about each section of the article, described below.\nThe strongest concern is the interpretation of compliance with the NICE guidelines, thus generating conclusions that do not align with actual findings. This is described in the ‘Results’ section. Based on these concerns, I cannot recommend this article for indexing in its current state.\nOverall\nPlease use person-first language throughout the article when describing obesity i.e. ‘people/person/child with obesity’, rather than ‘obese people/person/child’.\nTitle\nStarting with “reducing childhood obesity” suggests that the article describes an initiative that reduces prevalence. Please amend this to reflect the study purpose and/or results. Suggest using ‘Routine service evaluation of an early years…’\n\nIf using the word ‘borough’ in the title, suggest defining borough in the introduction or methods.\nIntroduction\nPlease describe the Early Years Nutrition Programme in more detail so the reader can understand its components. Is it consistent across the borough? How flexible is it? Who owns/controls its implementation?\n\nA statistic to support the statement ‘ethnically diverse population’ would be helpful.\n\nPlease add a prevalence (%) statistic to support Newham’s childhood obesity status.\n\nThe reference given for the Newham Childhood Obesity Action plan is incorrect.\n\nIt’s unclear in the study aim which programme is being assessed – please amend.\n\nIt is unclear if the Early Years Nutrition Programme informed Newham’s Action Plan or vice-versa – please clarify.\n\nWhat is the research question your study aimed to answer?\nMethods\nSubheadings would be helpful to structure the methods.\n\nYou define the study objectives and an additional aim at the start of the methods section – suggest moving to the end of the introduction.\n\nHow did you approach the children’s centres for recruitment?\n\nPlease add which authors collected the study data.\n\nPlease reference the NICE guidelines.\n\nWhat was compliance defined as? Was there a method of classification used to rank compliance?\nResults\nIf possible, including figures (photos) of the display boards to support the results would strengthen, as well as help readers understand what information is presented to support ‘generic health and wellbeing’, ‘breastfeeding’ etc.\n\nPlease describe what was involved in the parent sessions – their content, duration, frequency, facilitation etc. A table would help here. Why did more than 1/3 of centres not offer any sessions?\n\nThe statement that the “Early Years Nutrition Programme is meeting the NICE recommendations for child nutrition, achieving all recommendations within the scope of this study” is incorrect and does not align with the study aim:\nWhy could the EYNP not be assessed for compliance with the NICE recommendations prior to its implementation in child centres?\n\nTable 2\nIts unclear which sections of this table are taken from the NICE guidelines and which are added by the authors – please clarify.\n\nIn many instances, the evidence provided by the authors does not support the recommended action by the NICE guidelines. Therefore, the author’s conclusions aren’t supported.\nTraining – availability of training does not translate to appropriate knowledge and skills as this is not measured.\n\nFolic acid – availability of supplementation does not translate to HP’s advising about suitable folic acid supplementation.\n\nHealthy Start – how was the Healthy Start Scheme promoted in all centres? Saying it was is not evidence.\nTwo mentions of stock lists in this box.\n\nLink Workers – evidence does not support the action.\n\nInfant formula – the evidence does not support the action. Providing breastfeeding training does not confirm if professionals undertook the training, nor provide sound advice to women.\n\nChild health promotion – numerous evidence examples do not align with actions (e.g. added sugar, weighing children).\n\nWhy were some NICE recommendations out of scope (Diet in pregnancy, obesity)? This is not explained in the paper and no justification is given as to their exclusion, nor there is any obvious reason that justifies their exclusion.\n\nSome evidence is provided by services unrelated to the EYNP – e.g. Salvation Army.\n\nSome evidence provided is only relevant to one or two children’s centres (identified in brackets) – the authors cannot conclude that the EYNP meets the NICE guidelines in all children’s centres.\nSuggest mapping evidence to each centre and aggregating this data to assess overall compliance\n\nDiscussion\nPlease use subheadings to structure the discussion and start by summarising the results of your study – this is currently done at the end of the discussion.\n\nNo data is provided to support the statement that staff training has resulted in benefits for children in the present study.\n\nSee the program of research by Prof Sarah Redsell around UK health visitors delivering a childhood obesity prediction tool to families to inform targeted preventive intervention.\n\nSome references are old (2010) – please update your references to be more contemporary.\n\nAn additional limitation is the lack of process evaluation to determine actual compliance of EYNP with the NICE guidelines.\nConclusion\n18 recommendations are mentioned here but 22 in the methods?\n\nWhy is staff training an issue when this was reported as evidence for multiple NICE recommendations? How did you determine it was an issue?\n\nMost of the conclusion information is recycled from the discussion – suggest shortening conclusion to 1-2 sentences and strengthening other parts of the article.\n\nWhat are the steps for future research?\nKey messages and recommendations:\nBe clear where it is that childhood obesity is rising – the UK? Borough? Globally?\n\nThe third key message is briefly discussed in the article but is not evaluated – suggest changing/removing.\nData availability\nI disagree with this statement. Data underlying the results comprise photographs and field notes and personal/subjective interpretations of the NICE recommendations.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "87859", "date": "17 Sep 2021", "name": "John J Reilly", "expertise": [ "Reviewer Expertise Childhood obesity aetiology", "prevention", "treatment", "consequences", "diagnosis." ], "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 This short report describes an evaluation of early child nutrition education information in an area of London with high levels of deprivation and childhood obesity. The authors have compared the content of material displayed in 8 childcare centres against content recommended by NICE England.\nWhile the work is presented as an evaluation of childhood obesity efforts in the centres, the guidelines from NICE, and the topics addressed in the evaluation have relatively little relevance to obesity and refer largely to general nutrition/parenting.\nI struggled to follow what was done and how it was done (possibly the result of constraints imposed by the format), but assessment of content of materials on display is quite a limited assessment of the potential content/impact of what was on offer (the authors do acknowledge that in a well considered Discussion, section), and the limited scope of the work means that generalisability is quite likely to be limited.\nSpecific Comments\nTitle I see very little relevance to childhood obesity in the report and don't feel it is appropriate in the title.\nIntroduction The statement that childhood overweight and/or obesity in the UK has been on the rise in the UK is not consistent with other evidence and the evidence cited for it not clear. Overall prevalence has been relatively stable since around 2010, but with increasing prevalence in some sub-groups. The authors should check their statement / amend / clarify if necessary.\nSince the content of the paper is almost entirely about early childhood nutrition I think the Introduction should reflect that, rather than the focus on obesity. At the moment the Introduction and rest of the paper are quite disconnected.\nMethods While I realise that word limit makes a description of what was done challenging I still think that the Methods section needs to be clearer about what was done.\nI was quite confused by the different terms / concepts (standards / recommendations / guidelines) - are these the same thing? A clearer statement of exactly what the centre materials were being compared against would be helpful - Table 2 helps with this to some extent but the text needs to be clearer/more informative if it is to be understood easily by most readers.\nThe assessment of materials on display is in itself useful but quite a limited way of assessing implementation. While the authors acknowledge this the fact remains that as an evaluation of implementation what was done is only slightly informative.\nResults and Discussion I don't think the childcare centres should be identifiable (Table 1).\nThe content of table 2 is helpful in identifying what NICE recommended, but also tends to confirm that this isn't a set of recommendations that has very much to say about childhood obesity. The Introduction and Discussion should have a focus on early childhood nutrition more generally as that appears to be what the guidelines refer to.\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? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/9-682
https://f1000research.com/articles/9-680/v1	07 Jul 20	{ "type": "Systematic Review", "title": "Malaria vaccines targeting the pre-erythrocytic stage: a scoping review", "authors": [ "Teresa Ogeto", "Ferdinand Ndubi", "Mary Murithi", "Richard Kagia", "Esbon Wambugu", "Titus Suge", "Carolyne Chepkirui", "Josephat Tonui", "Fiona Maiyo", "Lydia Momanyi", "Michael Walekhwa", "Teresa Ogeto", "Ferdinand Ndubi", "Mary Murithi", "Richard Kagia", "Esbon Wambugu", "Titus Suge", "Carolyne Chepkirui", "Josephat Tonui", "Fiona Maiyo", "Lydia Momanyi" ], "abstract": "Malaria is a deadly infectious parasitic disease that causes devastating morbidity and mortality globally. Despite being a public health concern, an effective vaccine for prevention of the disease remains elusive. Global efforts are exploring possible ways of developing and improving vaccines to counteract the complex nature in which Plasmodium falciparum evades the immune system. A number of vaccines have been developed in the past targeting the various parasitic life cycle stages. Transmission blocker vaccines, such as PpPf S25, target the parasite stages in the mosquito vector. However, these herd vaccines only protect the immunized population. Vaccines targeting blood-stage forms, such as the AMA-1 and MSP-1 vaccines, are challenged by the complex metabolic pathways of erythrocytes and merozoites. Vaccines targeting the pre-erythrocytic sporozoite stage remain the most promising approach thus far. Here, we systematically review the literature on pre-erythrocytic stage vaccines and on-going work in the field. Furthermore, we highlight gaps in current knowledge and point to potential areas of future work. Articles on pre-erythrocytic malaria vaccines were obtained from Google scholar, PubMed and Cochrane starting from the year 2010. Ten papers were reviewed. A number of vaccines were reviewed highlighting; the vaccine type, clinical phase of trial, population demographics, vaccine immunogenicity, efficacy and safety. The RTS,S vaccine is reportedly the most advanced, having been rolled out for phase III clinical trials in a number of malaria-endemic African countries. The pre-erythrocytic vaccines discussed have made milestones in clinical trials. Some of the challenges elicited may be addressed via screening for novel antigens, exploring suitable vaccine administration vehicles, as well as using a combined multi-stage vaccine approach.", "keywords": [ "Malaria", "Pre-erythrocytic", "Plasmodium falciparum", "Vaccines", "Sporozoites", "efficacy" ], "content": "Introduction\n\nMalaria, a disease commonly found in the tropics and subtropics is caused by parasites of the genus Plasmodium whereby P. falciparum causes the greatest disease burden. In 2018, it was estimated to have caused 228 million cases globally, out of which 93% (213 million) occurred in Africa1. In Kenya, malaria is a major public health concern causing 6.7 million cases and 4000 mortalities in children annually2,3. Various community and facility interventional strategies, such as insecticide treated mosquito nets, indoor residual spraying, repellents and chemotherapy have significantly reduced the disease burden over the last decade4. However, these strategies are limited in their efficacy; hence elimination of the disease is unlikely without an effective vaccine5. Consequently, an effective vaccine conferring high protective efficacy is required to combat the disease.\n\nTo date, a number of vaccines have been developed and are at various stages of clinical trials. These vaccines target specific stage(s) of the parasite life cycle, such as pre-erythrocytic (liver-stage), erythrocytic (blood-stage) and gametocytes6. The pre-erythrocytic vaccines (RTS,S, PfSPZ, TRAP, PP) that target the sporozoites have proved to be the most efficacious of these7. Their superiority is founded primarily on the premise that the sporozoites harbour less antigens and are thus easily targeted by an appropriate vaccine. This results in reduced merozoite production thereby rendering the blood stage immune responses superior8. The pre-erythrocytic stage is however faced with the challenge of culturing sporozoites in vitro, making it difficult to explore novel antigens9.\n\nThe RTS,S vaccine, which has progressed to phase III clinical trials, has reportedly been found to be safe and immunogenic when tested in African children in malaria endemic regions such as Kenya and Tanzania10,11. However, trials of this vaccine done in Africa have revealed low efficacy levels. A study done by Olotu et al (2016), on children aged 5–17 months immunized with RTS,S/AS01A vaccine (cases) and rabies vaccine (controls), showed efficacy of 27% against first episode of clinical malaria11. Radiation-attenuated sporozoites (RAS) delivered through the bite of an infected mosquito was 100% protective in humans9. An irradiated P. falciparum sporozoites (PfSPZ) vaccine administered via intravenous injection also resulted in 100% protection in humans12. Other pre-erythrocytic vaccines that have so far been tested in humans include the thrombospondin related anonymous protein (TRAP), multi-epitope thrombospondin related adhesion protein (ME-TRAP) and polyprotein (PP) vaccines13,14.\n\nDespite the numerous interventions towards eliminating malaria, the parasite remains elusive. Vaccines remain to be the only hope to combat the disease. Therefore, efforts to understand P. falciparum biology and cross-talk mechanisms repressing the host immune response to an active infection are continuously being sought. In this review, we focus on milestones made on the pre-erythrocytic malaria vaccines in the last decade. We further highlight the challenges, immunogenicity and efficacy of these vaccines. Studies included mainly the RTS,S vaccine whose clinical trials were carried out in Africa, a malaria endemic region.\n\n\nMethods\n\nThis review was done according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA)15. Articles included were those that had the relevance of the subject of malaria vaccine.\n\nGoogle Scholar, Cochrane and PubMed were the sources of articles. The search terminologies used were ‘pre-erythrocytic malaria vaccines’ and ‘malaria vaccines’. An advanced search strategy in Google Scholar was done to return articles with all the words ‘pre-erythrocytic’ and ‘malaria vaccines’ in their titles. The exact phrases captured in the article titles were ‘pre-erythrocytic malaria vaccines’ or ‘malaria vaccines’. Article titles without the words ‘review’ or ‘meta-analysis’ or ‘expert opinion’ or ‘models’ dated between 2010–2020 were retrieved. The last search in the databases was done on 24th April 2020.\n\nClinical trials were the only article type considered; reviews, meta-analysis, expert opinions, in vitro studies, mathematical and computer modeling studies were excluded. Articles published in English in the last ten years (2010–2020) were considered since promising milestones have been achieved in the last 10 years.\n\nTo identify the suitability of the article to be included, the titles, abstracts, year of publication and type of study were considered. The papers were screened for eligibility by two reviewers and disagreements were resolved by consensus.\n\nRelevant data was extracted from the included studies based on the type of the vaccine, study design, the clinical trial phase, the mode of vaccine administration, efficacy and safety profile, as well as related adverse events. Other important relevant information, such as the experimental procedures, were also evaluated for each study.\n\n\nResults\n\nIn total, 17 articles were obtained from Google scholar, 7 from Cochrane and 231 from PubMed. After duplicate removal and screening, 10 articles were considered as meeting the criteria (Figure 1).\n\nTable 1 exhibits the characteristics of the 10 articles that were included.\n\n\nDiscussion\n\nThe RTS,S vaccine is by far the most advanced malaria vaccine in clinical development today. The vaccine confers its protective effect by targeting the circumsporozoite protein (CSP), a specific protein expressed by parasites in the pre-erythrocytic stage of the Plasmodium falciparum (Pf) human life cycle8. In specific field trials, RTS,S-containing vaccines have been administered with either the AS01 or AS02 adjuvant systems8. By virtue of their intrinsic properties and composition, therefore, vaccines containing the RTS,S formulation impart partial pre-erythrocytic immunity which is distinct from naturally-acquired immunity that mainly targets blood-stage parasites8,16. The latter type of immunity is hypothesized to be mediated mainly by T- and B-cell responses, as well as other possible mechanisms that are not presently well defined. An immuno-epidemiological study conducted by Bejon et al in 2011 concluded that vaccination of young children with the RTS,S/AS01E vaccine resulted in reduced exposure to blood-stage parasites, therefore reducing anti-merozoite antigen antibody concentrations. The antibodies were, however, not correlates of clinical immunity to malaria8. This is a serious concern associated with use of the RTS,S vaccine in the general population.\n\nSeveral attempts have been made to improve the protective efficacy of the RTS,S vaccines. These include, but are not limited to, formulation with more potent adjuvant systems, use of boosted regimens with other CSP-expressing regimens and evaluation of other Pf antigens separately or in combination with RTS,S. An example of such a study was conducted using RTS,S combined with a recombinant form of thrombospondin related anonymous protein (TRAP) of Pf (PfTRAP)18. Measurement of antigen-specific antibody responses, lymphoproliferative responses and IFN production due to the combination vaccine were similar to those produced by each of the single component vaccines. However, both the phase 1 and 2 clinical studies conducted by the investigators indicated inferior protective efficacy with the RTS,S/TRAP combination compared to single RTS,S/AS02 vaccine18. This was attributed to possible immunological interference, a phenomenon whereby multiple antigens interfere with T-cell priming and restimulation expected with a single antigen. The study was also limited by its significantly small sample size. Future combinations should perform preliminary exclusion of possible immunological interference between component antigens to improve success rates of such ‘boosted’ regimens.\n\nAn important consideration about use of the RTS,S vaccines is their duration of protective effect in the general population. Field trials of the vaccine have been conducted on infants and young children, where the vaccine has demonstrated average protection rates of between 18–36% with 3–4 doses of the vaccine administered during a 48-month follow-up period11. These trials were conducted in different African sites with varying rates of malaria transmission. Follow-up studies spanning a period of seven years however produced disappointing results on the long-term protective efficacy of the RTS,S malaria vaccine. From the two clinical studies, it was concluded that a three-dose vaccination regimen with RTS,S/AS01 vaccine offered initial protection against clinical malaria. The result was however offset in subsequent years, especially in areas with higher-than-average exposure to the parasites. Also, anti-merozoite antibody levels in vaccinated individuals were poor correlates of clinical immunity11.\n\nA recent study investigating the relationship between protective efficacy of the RTS,S/AS01 malaria vaccine and CSP polymorphisms in localized parasite population genotypes indicated that protective efficacy of the vaccine was higher in malaria parasites with matched CSP alleles than those with unmatched alleles16. This finding is important because CSP polymorphisms and differences in haplotypic regions of the protein exert an effect of vaccine efficacy16. Significant differences were observed in the cumulative vaccine efficiency rate between different age groups, based on the CSP polymorphisms. Therefore, it may be helpful to incorporate CSP polymorphisms in the local parasite population in future vaccine development efforts. Such an expensive venture would definitely impact on the cost of production, and availability, of these tailor-made malaria vaccines.\n\nOn the bright side, a study investigating the immunogenicity and safety of commercially produced RTS,S/AS01 malaria vaccine on a lot-to-lot basis yielded encouraging results. No significant variations were noted between the RTS,S/AS01 vaccine lots formulated from a commercial-scale purified antigen bulk batch compared to those formulated from a pilot-scale antigen bulk batch19. This is an encouraging prospect for future production of multiple batches of the RTS,S pre-erythrocytic vaccines especially in resource-limited settings which are also malaria endemic.\n\nA different approach from the RTS,S vaccine adopted in the last decade has led to development of the Pf sporozoite (PfSPZ) vaccine12. This vaccine contains radiation-attenuated sporozoites of the parasite, and was assessed for tolerability, safety, immunogenicity and protective efficacy. The PfSPZ vaccine was administered by direct venous inoculation of 3–5 doses in non-immune individuals12. The PfSPZ vaccine, given in a three-dose regimen, protected against both 3- and 24-week homologous (similar strain as in vaccine) controlled malaria infections. No significantly alarming side effects were associated with the trialed regimens of this vaccine, and this is an encouraging outcome for future applications of the PfSPZ vaccine in mass malaria vaccination campaigns. Upcoming phase III trials of the vaccine will provide further insights on the protective efficacy and safety of the vaccine, especially against heterologous (different strain from vaccine) infections.\n\nFurthermore, a polyprotein (PP) pre-erythrocytic malaria vaccine containing the entire sequence of six separate Pf proteins has been investigated. This vaccine was delivered using the combined viral vectors fowl pox virus strain FP9 and modified vaccinia virus Ankara (MVA)14. The design strategy of this ‘polyprotein’ vaccine was targeted at producing a broad antibody response against a broad range of plasmodial pre-erythrocytic antigens, as opposed to a strong but narrow response. No serious adverse effects were observed with both the FP9-PP and MVA-PP vaccines, but both failed to induce protection against sporozoite challenge or delay onset of parasitemia in vaccinated individuals14. The investigators attributed this outcome to possible limits in the size of immunogenic pre-erythrocytic proteins, at least when attached to viral vectors. Future work in the design of PP pre-erythrocytic malaria vaccines should therefore attempt to investigate the theory of size limits to immunogenicity, as well as effects of specific combinations and sizes to efficiency of the viral vectors employed. In addition, a separate phase IIa study utilizing the chimpanzee adenovirus 63 (ChAd63) and MVA vectors to express a TRAP-based vaccine yielded more promising results13. Both vaccines were demonstrated to be safe and adequately immunogenic, but induced only moderate T-cell response. This immunogenic response was not sufficient to provide protection against clinical malaria in the follow-up period. Despite these results, viral vectors have a role in malaria vaccine delivery and future work should further investigate novel viral vectors as well as increased applicability in design of pre-erythrocytic vaccines.\n\nAlthough CSP has stood out over the last few years as an immuno-dominant pre-erythrocytic antigen, several other proteins can be utilized in the design of pre-erythrocytic malaria vaccines. A study of 27 different pre-erythrocytic antigens indicated that 21 out of those, all localized to different parts of the sporozoite, induced detectable antibody responses in animal models9. These antigens elicited adequate antibody and T-cell responses and are therefore possible vaccine candidates. In future development of pre-erythrocytic malaria vaccines, thorough investigation of these additional sporozoite proteins should be undertaken to explore their viability as starting points for vaccine development. It would be interesting to explore these additional proteins as vaccine candidates.\n\nThis review had some limitations. We only considered vaccine candidates in clinical trials in the past decade. Therefore, promising vaccine candidates which have not yet been approved for clinical trials were not considered. In addition, only the vaccine candidates affecting the pre-erythrocytic stage were evaluated yet it is possible for a molecule affecting the erythrocytic stage to be an effective malaria vaccine. Articles published in another language apart from English were not included leading to exemption of potentially promising vaccine candidates.\n\n\nConclusion\n\nThe search for a malaria vaccine targeting the pre-erythrocytic stage of the Pf has led to development of sub-unit vaccines, for example, RTS,S vaccine & ChAd63 MVA METRAP vaccine and inactivated vaccines like radiation attenuated Pf sporozoite vaccine. However, these vaccines have not been shown to induce sufficient immune responses required to provide adequate protection against malaria. The RTS,S vaccine currently undergoing clinical trials remains the most promising. However, its inability to confer long-term protection against clinical malaria and the varied antibody responses associated with the vaccine remain major drawbacks. The attenuated sporozoite vaccine is also a promising option, and ongoing clinical trials are vital in providing conclusions on the protective effect of this vaccine. Most importantly, ability of the PfSPZ vaccine to impart protection against heterologous malaria infections will be vital. Use of viral vectors for malaria vaccines has proven to be a viable mode of delivery. Further work is needed to identify optimal viral vectors for maximum protective efficacy. In addition, the possibility of using combined antigens in a single vaccine and further exploration of other plasmodial antigens as vaccine candidates are both routes that need to be pursued.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nHarvard Dataverse: PRISMA-ScR checklist for ‘Malaria vaccines targeting the pre-erythrocytic stage: a systematic review’, https://doi.org/10.7910/DVN/ZK3ZZH20.", "appendix": "Acknowledgements\n\nWe acknowledge the technical input of Ms. Regina Njeru of the International Livestock Research Institute (ILRI). We are very grateful that she made time to give her valuable contribution.\n\n\nReferences\n\nWorld Health Organization World Malaria Report: World Health. WHO/HTM/GM, 2010; 238. Reference Source\n\nSultana M, Sheikh N, Mahumud RA, et al.: Prevalence and associated determinants of malaria parasites among Kenyan children. Trop Med Health. 2017; 45: 25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHalliday KE, Okello G, Turner EL, et al.: Impact of Intermittent Screening and Treatment for Malaria among School Children in Kenya: A Cluster Randomised Trial. PLoS Med. 2014; 11(1): e1001594. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWest PA, Protopopoff N, Wright A, et al.: Indoor Residual Spraying in Combination with Insecticide-Treated Nets Compared to Insecticide-Treated Nets Alone for Protection against Malaria: A Cluster Randomised Trial in Tanzania. PLoS Med. 2014; 11(4): e1001630. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCoelho CH, Doritchamou JYA, Zaidi I, et al.: Advances in malaria vaccine development: Report from the 2017 malaria vaccine symposium. npj Vaccines. 2017; 2: 34. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCrompton PD, Pierce SK, Miller LH: Advances and challenges in malaria vaccine development. J Clin Invest. 2010; 120(12): 4168–78. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDuffy PE, Sahu T, Akue A, et al.: Pre-erythrocytic malaria vaccines: identifying the targets. Expert Rev Vaccines. 2012; 11(10): 1261–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBejon P, Cook J, Bergmann-Leitner E, et al.: Effect of the pre-erythrocytic candidate malaria vaccine RTS,S/AS01E on blood stage immunity in young children. J Infect Dis. 2011; 204(1): 9–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAguiar JC, Bolton J, Wanga J, et al.: Discovery of novel plasmodium falciparum pre-erythrocytic antigens for vaccine development. PLoS One. 2015; 10(8): e0136109. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNdungu FM, Mwacharo J, Wambua J, et al.: A seven-year study on the effect of the pre-erythrocytic malaria vaccine candidate RTS,S/AS01E on blood stage immunity in young kenyan children [version 1; peer review: 1 approved, 2 approved with reservations]. Wellcome Open Res. 2019; 4: 42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOlotu A, Fegan G, Wambua J: Seven-Year Efficacy of RTS,S/AS01 Malaria Vaccine Among Young African Children N Engl J Med. 2016; 374(26): 2519–29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEpstein JE, Paolino KM, Richie TL, et al.: Protection against Plasmodium falciparum malaria by PfSPZ Vaccine. JCI Insight. 2017; 2(1): 1–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTiono AB, Nébié I, Anagnostou N, et al.: First field efficacy trial of the ChAd63 MVA ME-TRAP vectored malaria vaccine candidate in 5-17 months old infants and children. PLoS One. 2018; 13(12): e0208328. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPorter DW, Thompson FM, Berthoud TK, et al.: A human phase I/IIa malaria challenge trial of a polyprotein malaria vaccine. Vaccine. 2011; 29(43): 7514–22. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShamseer L, Moher D, Clarke M, et al.: Preferred reporting items for systematic review and meta-analysis protocols (prisma-p) 2015: Elaboration and explanation. BMJ. 2015; 350: g7647. PubMed Abstract \| Publisher Full Text\n\nNeafsey DE, Juraska M, Bedford T, et al.: Genetic diversity and protective efficacy of the RTS,S/AS01 malaria vaccine. N Engl J Med. 2015; 373: 2025–2037. Publisher Full Text\n\nAgnandji ST, Lell B, Soulanoudjingar SS, et al.: First results of phase 3 trial of RTS,S/AS01 malaria vaccine in African children. N Engl J Med. 2011; 365(20): 1863–1875. PubMed Abstract \| Publisher Full Text\n\nKester KE, Gray Heppner D, Moris P, et al.: Sequential Phase 1 and Phase 2 randomized, controlled trials of the safety, immunogenicity and efficacy of combined pre-erythrocytic vaccine antigens RTS,S and TRAP formulated with AS02 Adjuvant System in healthy, malaria naïve adults. Vaccine. 2014; 32(49): 6683–6691. PubMed Abstract \| Publisher Full Text\n\nUmeh R, Oguche S, Oguonu T, et al.: Immunogenicity and safety of the candidate RTS,S/AS01 vaccine in young Nigerian children: A randomized, double-blind, lot-to-lot consistency trial. Vaccine. 2014; 32(48): 6556–6562. PubMed Abstract \| \|Publisher Full Text\n\nWalekhwa M: Malaria vaccines targeting the pre-erythrocytic stage: a systematic review. Harvard Dataverse, V3.2020. Reference Source" }	[ { "id": "71931", "date": "26 Oct 2020", "name": "James M Burns Jr", "expertise": [ "Reviewer Expertise malaria vaccines" ], "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 manuscript, Ogeto et al attempt to provide a comprehensive review of malaria vaccines targeting pre-erythrocytic stages of Plasmodium falciparum. The review falls short of achieving that goal. Although the period from 2010-2020 was considered, only 10 publications were evaluated. The criteria for selection of these articles was not adequately presented. For example 155 articles were deemed not eligible by two reviewers, but the basis for exclusion was not clear. The bulk of the article is Table 1 which simply provides a basic description of each study and a list of findings. In some cases, the information is not adequate to understand the composition of each vaccine formulation and/or to appreciate the primary goal of each study. Overall, the review lacked depth, breadth and a clear theme for an integrated comparison of the papers selected. Considering published reviews covering related topics (i.e. progress on malaria vaccine development, RTS,S, whole sporozoite vaccines), there was little new information or insight provided by the authors. The effort could benefit from a more thorough, critical evaluation of clinical trial results. It is not clear that this summary of pre-erythrocytic malaria vaccine trials will be of substantive value to the field.\n\nIn places, the point the authors were trying to convey was not clear. For example, the basis for the following statements is not apparent and/or is confusing:\n\nAbstract: “Vaccines targeting blood-stage forms, such as AMA-1 and MSP-1 vaccines, are challenged by the complex pathways of erythrocytes and merozoites.”\n\nIntroduction: “Their superiority is founded primarily on the premise that sporozoites harbor less antigens and are thus easily targeted by an appropriate vaccine. This results in reduced merozoite production thereby rendering the blood stage immune responses superior”.\n\nAre the rationale for, and objectives of, the Systematic Review clearly stated? Yes\n\nAre sufficient details of the methods and analysis provided to allow replication by others? No\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? No", "responses": [] }, { "id": "96331", "date": "07 Oct 2021", "name": "Miguel Prudêncio", "expertise": [ "Reviewer Expertise Pre-erythrocytic stage of Plasmodium infection", "Malaria vaccines", "Antiplasmodial therapeutics" ], "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 set out to write a “scoping review” of pre-erythrocytic malaria vaccines. However, this manuscript does not even remotely cover all relevant aspects of pre-erythrocytic vaccination against malaria. This is hardly surprising, when the authors themselves claim to have reviewed “ten papers” to write this review, when there are hundreds of papers on this subject, and many dozens of these include results on the clinical evaluation of pre-erythrocytic malaria vaccine candidates. It is not even clear how these ten papers were selected, let alone the fact that it is very arguable that they are all among the most relevant in the field. For example, there are no references to genetically attenuated sporozoites (GAS), sporozoites administered under chemoprophylaxis (CPS), Sanaria's PfSPZ-CVAC, a newly described strategy based on genetically modified P. berghei parasites expressing P. falciparum antigens, or the new subunit vaccine candidate R21 developed by the University of Oxford, all of which have been tested in the clinic. This places this manuscript a very long way from being considered \"a scoping review\".\nBesides the evident absence of many references to crucial work in this field, the manuscript is also fraught with incorrections or misleading remarks, including in the abstract, such as “transmission blocker (sic) vaccines … only protect the immunized population” (vaccines usually aim to protect those immunized; what the authors probably meant is that transmission-blocking vaccines are altruistic vaccines that only work at the community level) and “Vaccines targeting blood-stage forms… are challenged by the complex metabolic pathways of erythrocytes and merozoites” (I would argue that the challenges for blood stage vaccines go well beyond the metabolism of erythrocytes and merozoites). Overall, the article falls very short of its stated objective and, in my opinion, does not meet the minimal standards for indexing.\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? No\n\nIs the statistical analysis and its interpretation appropriate? Not applicable\n\nAre the conclusions drawn adequately supported by the results presented in the review? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-680
https://f1000research.com/articles/8-271/v1	11 Mar 19	{ "type": "Research Article", "title": "Toward a paradigm shift from deficit-based to proactive speech and language treatment: Randomized pilot trial of the Babble Boot Camp in infants with classic galactosemia", "authors": [ "Beate Peter", "Nancy Potter", "Jennifer Davis", "Inbal Donenfeld-Peled", "Lizbeth Finestack", "Carol Stoel-Gammon", "Kari Lien", "Laurel Bruce", "Caitlin Vose", "Linda Eng", "Hanako Yokoyama", "Daniel Olds", "Mark VanDam", "Nancy Potter", "Jennifer Davis", "Inbal Donenfeld-Peled", "Lizbeth Finestack", "Carol Stoel-Gammon", "Kari Lien", "Laurel Bruce", "Caitlin Vose", "Linda Eng", "Hanako Yokoyama", "Daniel Olds", "Mark VanDam" ], "abstract": "Background: Speech or language therapy is typically initiated reactively after a child starts showing delays. Infants with classic galactosemia (CG), an inborn error of metabolism with a known high risk for both speech and language disorders, hold the keys towards evaluating whether preventive treatment is effective when the risks are known at birth. We present pilot data from a randomized parallel trial of an innovative proactive speech and language intervention program, the Babble Boot Camp (BBC). Method: Five children with CG, otherwise healthy, participated in the BBC from approximately 2 to 24 months of age. One of these was randomly selected as control receiving conventional management. A pediatric speech-language pathologist met weekly via telepractice with the parents in the treatment cohort. Parents implemented the prespeech, speech, and language stimulation and expansion activities according to the protocol. The control child was still too young for conventional treatment. Primary outcome measures were speech sound production complexity in babble and speech and expressive vocabulary size. Secondary outcome measures were developmental milestones in communication, motor, and cognition. Outcomes in the treatment cohort were compared to typical children and the control child. The trial is ongoing. Results: All four treated children had higher speech sound skills in babble, three had higher speech sound skills in meaningful speech, two had higher expressive vocabularies, and three had higher communication and personal-social skills, compared to the control child with CG. Discussion: Given the high risk for speech and language delays in children with CG, finding on-schedule abilities in two or more of the treated children but not the untreated child is unexpected under random conditions. The trends toward beneficial effects of the BBC on speech sound production, expressive language, and communication milestones warrant appropriately powered larger clinical trials with full randomization. Trial registration: ClinicalTrials.gov NCT03838016 (12th February 2019).", "keywords": [ "speech disorder", "language impairment", "genetic risk", "infant", "very early intervention", "prevention" ], "content": "Introduction\n\nDifficulties with speech and language are common among young children. In the US, 11% of children age 3 to 6 years have a communication disorder (Black et al., 2015). Many parents who are concerned about their child’s ability to talk ask the child’s doctor, who, in turn, may refer them to a speech-language pathologist. By the time a referral is made, the child may already be two, three, or even four years old and has passed critical stages in the process of speech and language development (Cates et al., 2012). There is strong evidence that early interventions in children with known risks or first signs of a variety of disorders are highly effective, for instance interventions for children with autism spectrum disorder as young as 12 months (Dawson et al., 2010; Guralnick, 2011; Rogers et al., 2014). However, very early speech and language services are not yet available, in part because speech and language are later-developing skills and disorders in these areas cannot be reliably diagnosed on behavioral grounds until an age when deficits become evident, greater than 24 months for speech (Goldman & Fristoe, 2015) and greater than 36 months for language (Semel et al., 2004).\n\nThere are some early red flags during infancy that may foreshadow a later diagnosis of speech sound disorder and language impairment. Examples are sparse and delayed babbling (American Speech-Language-Hearing Association, 2007; DePaolis et al., 2013; McCune & Vihman, 2001; Oller et al., 1998; Oller et al., 1999; Stoel-Gammon, 1989; Stoel-Gammon, 2011; Vihman & Greenlee, 1987; Vihman et al., 1985), low engagement in activities with joint attention (Mundy et al., 2007), and failure to use gestures (Rowe & Goldin-Meadow, 2009). Specifically regarding babbling, a close predictive link between quantity/quality of babble and speech and language development later on has been previously described in typical children (American Speech-Language-Hearing Association, 2007; DePaolis et al., 2013; McCune & Vihman, 2001; Oller et al., 1998; Oller et al., 1999; Stoel-Gammon, 1989; Stoel-Gammon, 2011; Vihman & Greenlee, 1987; Vihman et al., 1985). However, whether treatment targeting these early signs of communication has a beneficial effect on later speech and language development is unknown because such treatments have not been developed and validated. Going even further back in the developmental trajectory, some children are born with risk factors for speech and language disorders and this risk is known long before prespeech behaviors such as coo and babble and actual speech and language emerge. The question is whether proactive, preventive treatment, if it existed, could reduce the deleterious effects associated with the disease in these cases and thereby improve outcomes.\n\nInfants with classic galactosemia (CG) are an ideal population to investigate whether proactive interventions during the first two years of life, long before traditional assessment and intervention are available, can significantly improve speech and language outcomes. CG is a recessively inherited inborn error of metabolism diagnosed via newborn screening, with incidence rates in the US ranging from 1/30,000 to 1/60,000. Newborn diagnosis can be life saving because of the deleterious effects of galactose buildup in the child’s blood that can occur if dietary restrictions are not implemented immediately. Despite rigorous dietary management, however, children with this disease have a substantially higher risk, compared to the typically developing (TD) population, not only for motor and learning disabilities (Antshel et al., 2004; Karadag et al., 2013; Potter et al., 2013) but also, importantly, for severe speech and language disorders. Speech disorders were reported in 77% of children with CG (Hughes et al., 2009), compared to 3.8% among children generally (Shriberg et al., 1999), and language impairment in 90% of children with CG (Waggoner et al., 1990), compared to 7.4% among children generally (Tomblin et al., 1997). This elevated risk, coupled with the early identification, makes the CG population ideal to examine the efficacy of prospective intervention therapy. If proactive intervention is shown to be more effective than conventional management, this has the potential to change the management model from deficit-based to preventive services for these infants. It will also motivate similar studies in infants with other types of risk for communication disorders, for instance very low birth weight and 7q11.23 duplication syndrome. That is, children known to be at risk may benefit from early, prospective intervention, thus improving outcomes.\n\nThe Babble Boot Camp (BBC) is a program of activities and routines designed for children during the pre-speech and very early speech and language stages. It contains components intended to shape dyadic interactions across modalities, stimulate earliest vocalizations (coo, babble), support emergence of first words and sentences, and foster vocabulary and syntax growth. The active phase of intervention covers ages 2 to 24 months, with plans for follow-up testing using a professional evaluation of speech, language, and cognitive abilities at ages 36 and 48 months.\n\nHere, we report pilot results of the BBC. This Phase 0 exploratory study demonstrates, with clear clinical application, a viable proactive early intervention approach for minimizing speech and language disorders in a vulnerable population of infants with a known genetic risk for these disorders. The purpose of this pilot study was to examine if children with CG participating in the BBC show age-expected markers of expressive speech and language and to compare their outcomes to those in the control child. The primary focus is speech and language development measured with standardized assessments, with secondary attention to cognitive and motor development.\n\n\nMethods\n\nThis study was conducted with approval of the Institutional Review Boards at Arizona State University (IRB ID # STUDY00004969) and Washington State University (IRB ID # 13099). It began on January 31, 2017, and the trial is ongoing. Parents learned about the study through online research announcements and referrals from physicians or other service providers and contacted the research team. Once eligibility for participation was established and parents made the decision to participate, they gave written permission for their infants’ participation and written consent for their own participation. The study is listed on ClinicalTrials.gov under NCT03838016.\n\nThe current participants are 14 children with CG and their parents. Here, we report on a subset of the children, the five oldest children with the longest participation record, for whom a nearly complete dataset is available through age 21 months. This pilot treatment cohort consists of four children, two girls (codes CG1, CG2) and two boys (CG4, CG5). Note that CG1 only participated in the study up to age 18 months, due to personal circumstances. One additional boy with CG was randomly selected to serve as a control who did not participate in the BBC treatment program. All families participated in the close monitoring components of the study, described further below. In the near future, this parallel design will be built out by recruiting more children into the treatment and control cohorts using randomization and blinding, the latter of which can only be applied to research team members who analyze the data.\n\nIn- and exclusionary criteria are identical for the treatment and control children with CG and are designed to evaluate the effects of the treatment while keeping all other factors the same. Age at entry into the study is approximately 2 months. All infants are required to have a newborn diagnosis of CG. Infants with additional health or sensory diagnoses are excluded. Boys and girls of any racial/ethnic background are equally eligible to participate. Primary language in the home must be English and at least one parent must have at least an 8th grade education. Because the intervention is implemented via telepractice software, any family whose primary language is English can participate, regardless of country of residence. The cohort as a whole currently includes families in the US and the UK.\n\nThe BBC is implemented via parent training by a speech-language pathologist (SLP) with expertise in early childhood. The SLP uses a HIPAA-compliant telepractice computer interface to connect with the families. Parents learn about the typical milestones of prespeech, speech, and language development, potential red flags for delays, and, importantly, specific activities that support typical development for all stages of the program and development beyond.\n\nFollowing an orientation to the program, the SLP meets with each family once per week for approximately 10 minutes to train and consult on the relevant activities given the child’s current speech and language status. Examples of activities are stimulating and reinforcing coos and babble, enriching the child’s linguistic environment with joint book reading (Storkel et al., 2017), pointing out the names of objects (Dimitrova et al., 2016), and expanding child utterances to provide slightly more complex model sentences (Hassink & Leonard, 2010). Activities and routines are modeled on typical developmental milestones for ages 2 to 24 months reported in the scientific literature (Chapman, 2000; Gordon-Brannan & Weiss, 2007; MacLeod, 2013; Paul & Norbury, 2012, Stoel-Gammon, 1983; Stoel-Gammon, 2011). Children in the BBC treatment cohort progress through these milestones not based on age but on present levels of ability.\n\nOne key principle underlying all activities is the zone of proximal development or scaffolding in which parents provide speech and language models that bridge what the child can already do and what is slightly beyond the child’s skill set: the model is in the zone of skills that the child can do with help (Vygotsky, 1979). One key skill that is targeted throughout the program is imitation.\n\nBecause the BBC is designed to beneficially influence speech and language development, the primary outcome variables are speech sound production in babble and speech and expressive language ability. Secondary areas of interests are cognitive and motor development and quality of life, but these are only partially addressed in this report. During the active BBC phase, we monitor all of these areas as well as a range of other enrichment variables including volubility of child vocalizations, environmental influences, and demographic factors.\n\nSpecifically to assess speech sound development and language growth, we use a combination of several standardized tests and established clinical procedures. In the present pilot report, we quantify speech and language growth using several metrics. First, we focus on the complexity of the produced speech sounds during babble and early speech. Once per month per child starting at age 6 months, we compute the Mean Babbling Level (MBL) (Stoel-Gammon, 1989), a clinical measure of speech sound complexity for babbling. To compute the MBL, a set of at least 50 utterances is compiled and transcribed into the International Phonetic Alphabet. An expert rater assigns a score of 1, 2, or 3 to each utterance and computes the average; thus, MBL scores range from 1 to 3 for each child at any given time point. A score of 1 is assigned to simple utterances consisting of a vowel, a syllabic consonant, or a consonant-vowel (CV) or vowel-consonant (VC) sequence where the consonant is either a glide (“w”, “y”) or a glottal stop, defined as a brief silence interrupting a vowel; note that glottal stops and glides are not considered to be true consonants. Examples of level 1 utterances are “m” and “wawa”. A score of 2 is assigned to utterances containing at least one CV or VC sequence with a true consonant; if there are two or more syllables, the consonants may be the same ones or differ only in whether they are voiced or not. Examples are “bapa” and “dida”. A score of 3 is assigned to utterances containing at least two true consonants produced in different parts of the mouth and/or with different airflow characteristics. Examples are “gaba”, and “adap”. The three scores thus express the progression from motorically and linguistically simple to more complex skills. MBL scores in the BBC treatment and control cohort were compared to MBL scores in TD children at equivalent ages (Morris, 2010). Whereas the MBL is applied to babble, which does not convey lexical meaning, an equivalent measure called Syllable Structure Level (SSL) exists for meaningful speech (Paul & Jennings, 1992), and as for the MBL, we used this measure in tandem with typical norms (Morris, 2010). The source materials for the MBL and SSL are daylong naturalistic audio recordings captured with a passive, wearable audio recorder (LENA Research Foundation, Boulder, CO). The recordings are obtained in the natural environment of the families and children. The recorder is returned to the research labs and processed offline to obtain the raw, daylong audio file, which provided the source for the present MBL and SSL scores. Other measures based on the recordings will be reported in the future, including estimates of child volubility such as syllables per hour and vocalizations per hour, speech production and discourse characteristics of the target child and other family members, and other variables known to be important for language and communication development in children. All measures collected in this way are objective and algorithm-driven. For purposes of the present work, we extracted multiple 5-minute audio segments with the highest occurrence of child utterances in order to transcribe these utterances into the International Phonetic Alphabet and to compute MBL and SSL scores for each child and month.\n\nSecond, we use the MacArthur-Bates Communicative Development Inventories 2 (MBCDI-2) (Fenson et al., 2007) to capture early expressive and receptive vocabulary sizes as reported by the parents when filling out the MBCDI-2 protocol forms. MBCDI-2 questionnaires were collected from each family at regular intervals (ages 12, 15, 18, 21, and 24 months), and computed percentile scores were compared between the treated and untreated participants. Percentiles were based on the publisher’s norms, separately for boys and girls. Here, we focus on receptive vocabulary, available up to age 15 months, and expressive vocabulary, available for all reported ages but regarding our pilot sample, only through age 21 months.\n\nThird, the Ages and Stages Questionnaires - 3 (ASQ3) parent questionnaires (Squires & Bricker, 2009) capture communication abilities and personal-social development of young children. The evaluation of communication abilities differs from the expressive and receptive vocabulary checks in the MBCDI-2 in that it samples broader communication abilities, as relevant for child age, such as comprehending phrases, using language to achieve a goal, and producing multi-word sentences. The personal-social development component queries skills in activities of daily living such as feeding and dressing oneself and social interactions, both verbal and nonverbal, such as giving/receiving objects, asking for help, and role-play, as relevant for age. The ASQ3 is considered valid and reliable for purposes of tracking typical development in these and three other areas (gross motor, fine motor, and problem solving) and for identifying areas of concern, where the problem solving component is an estimate of cognitive ability (Schonhaut et al., 2013). All five questionnaire topics are applicable to children with CG because of the known risks for deficits in each of these areas (Antshel et al., 2004, Karadag et al., 2013, Potter et al., 2013). Each component is scored by summing the raw scores, then assigning one of three categories, based on the provided norms: Above cutoff (“On schedule”), close to cutoff (“Provide learning activities and monitor”), or below cutoff (“Assess further”). The ASQ3 questionnaires are available in 21 separate, age-appropriate and age-normed sets from ages 2 to 60 months. We administer this tool in 6-months intervals and report here on ages 12 and 18 months.\n\nResults and trends are presented descriptively, supported with graphs. Because of the small sample size of this pilot study, statistical tests for group differences and other inferential statistical procedures were not possible here, but will be used in future reports of the study.\n\nQuestionnaire data from the MBCDI-2 and ASQ3 were entered into the database by teams of at least two trained research assistants. The MBL and SSL scores were computed by three trained research assistants; 15% of their scores were double-scored by other team members, and differences of over 10% in a given child’s MBL score will be resolved by consensus; however, such a discrepancy has not occurred in the pilot data.\n\n\nResults\n\nMBL scores of the children with CG in the treatment cohort consistently exceeded those of the control child with CG and typical control children without CG (Figure 1). For the ages for which data were available for nearly all children (11 through 21 months), the average difference between the treatment cohort and the control child with CG was 1.01, indicating that the children with CG in the treatment cohort obtained substantially higher MBL scores than the children in the other two categories. For the ages for which data were available for the control child with CG and the typical control children without CG (12, 15, 16, 18, and 20 months, listed in Morris, 2010), the average difference was -0.04, indicating that the control child with CG obtained MBL scores roughly equivalent to the typical controls without CG, but note the declining trend for the control child at the most recent ages. The difference between the children in the treatment cohort and the typical children was -0.77, indicating that the children in the treatment cohort outpaced the typical children on average. Figure 1 shows MBL scores for all children and all available ages.\n\nSimilar to the MBL, the SSL is calculated on the basis of 50 utterances, but unlike the MBL, it is based on meaningful speech. For one child in the treatment cohort, CG1, not enough meaningful utterances could be identified in any of the recording sessions up to 18 months, when the child left the study, to compute the SSL. Of the remaining children and for the available ages at 12, 13, 15, 19, and 20 months, the children in the treatment cohort with CG outperformed the control child with CG by 1.0. For ages 15 and 19 months, the only ages for which data were available for the treatment cohort and the typical children without CG, the children in the treatment group outperformed the typical children by 0.6. The control child with CG and the typical children without CG could not be compared directly because data at the same ages were not available, but at ages 20 and 21 months, his scores appear lower than the typical controls’ scores at age 19 and 22 months. Note that, with only one exception, the highest scores were obtained by CG2 and CG5. Figure 2 summarizes the SSL scores.\n\nRegarding expressive vocabulary size, the control child obtained one of the two or three lowest rankings for the ages for which data were available (Figure 3). Two children in the treatment cohort, CG2 and CG5, obtained the highest percentile rankings, which, at age 21 months, were 73 and 72, respectively. Two children, CG1 and CG4, obtained low expressive vocabulary scores, with CG1’s percentile score being 1.7 at 18 months and CG4, 4.0 at 21 months. The control child, similar to CG1 and CG4 in the treatment cohort, showed declining expressive vocabulary scores, below the 8th percentile at 21 months. Note that not having any words at all at age 12 months, which was the case for CG1 and the control child, corresponds to the 25th percentile for boys and the 20th percentile for girls, but the ranking drops rapidly with age if the expressive vocabulary does not increase substantially.\n\nRegarding receptive vocabulary, the control child obtained the second-lowest percentile ranking (Figure 4), but note that all children including the control child show typical receptive vocabulary scores at the two ages represented in the questionnaires. The lowest percentile score, 10.5, is considered low average; it was obtained by CG1 at age 12 months. All other scores are solidly within normal limits. Figure 3 and Figure 4 summarize expressive and receptive vocabulary percentiles, respectively.\n\nThe AGS3 questionnaires at 12 and 18 months indicated that three of the four children in the treatment cohort had communication and personal-social abilities as expected for age. One child in this cohort had communication abilities below expectation for age at 18 months, and personal-social abilities close to the cutoff for age expectations. The control child scored close to the cutoff for age expectation at 18 months for communication, and at 12 and 18 months for personal-social abilities.\n\nRegarding problem solving, of the children in the treatment cohort, only CG1 and CG4 had scores that were not consistently in the range of scores expected for age, both at age 18 months, whereas the control child was close to the cutoff at both time points. Regarding fine and gross motor skills, CG1 was the only child in the treatment cohort with scores that were below (fine motor) or close to (gross motor) the cutoff, whereas the control child had scores close to the cutoff in both areas at one time point and, for gross motor, at both time points. Across all areas, mainly one child in the treatment cohort (CG1) and the control child did not show developmental skills on schedule. Table 1 summarizes the ASQ3 scores for all children in the five assessed areas.\n\nNote: AC = Above cutoff (on schedule), BC = Below cutoff (assess further), CC = Close to cutoff (monitor)\n\n\nDiscussion\n\nIn a small sample of very young children with a known and highly predictive risk for speech and language disorders due to CG, we show that a program of preventive activities and routines, the BBC, may have beneficial effects in three important regards.\n\nFirst, very early attention to speech sound production may increase children’s ability to produce more complex speech sounds in babble and speech. The children with CG in the treatment cohort obtained greater MBL scores during babble than the control child with CG as well as typical control children without CG. A similar pattern was seen for the SSL during meaningful speech. Together, these findings suggest that very early attention to speech sound production increases the ability to produce more complex speech sounds in babble and speech, in this case even beyond the expected levels of typically developing children. It is possible that MBL and SSL do not measure areas of crucial weakness in children with CG in general, but that targeting speech sound skills may give them a clear boost that may have beneficial effects on later speech and language development. Continued data collection and analysis in this population will provide conclusive evidence in this regard.\n\nSecond, the expressive vocabulary, and possibly to a lesser degree the receptive vocabulary, of children with CG may improve as a result of the BBC. Expressive vocabulary scores were completely within normal limits for two children in the treatment group, CG2 and CG5, whereas the control child with CG and two children in the treatment cohort, CG1 and CG4, scored below expectation. Given the 90% risk for language impairment in children with CG, finding two of four children in the treatment cohort with typical language skills may show the beneficial effects of the BBC program. The fact that receptive vocabulary scores were within normal limits for all children including the control child with CG may indicate that CG affects expressive language skills more than receptive language skills.\n\nThird, there may be gains in competence in using language and nonverbal means to interact with others. Three of the children in the treatment cohort had communication skills and personal-social skills as expected for age, as measured with the ASQ3. These children may be gaining age-appropriate competence in activities of daily living as well.\n\nComparing the results from all measures, the two children with the overall highest scores were CG2 and CG5. They showed no evidence of deficits in any of the evaluated area; in fact, in the areas of speech sound complexity in meaningful speech and expressive vocabulary size, they scored at the top of the pilot sample. By contrast, CG1 did not produce enough meaningful speech for SSL scores, had low expressive vocabulary scores, and scored near or below the cutoff for expectation in all five developmental domains assessed with the ASQ3. CG4 had intermediate MBL and SSL scores but low expressive vocabulary scores and one low score in the AGQ3, namely in problem solving. The control child obtained some of the lowest MBL, SSL, expressive vocabulary, and ASQ3 scores. These patterns are consistent with cross-domain associations among the evaluated skills (speech, language, social interaction, cognitive ability, motor ability). Whether these patterns reflect global levels of disease severity, global benefits from the treatment, or a combination of these cannot be ascertained based on the present data.\n\nBecause of the small sample size in this pilot study, generalizations to other children with CG are not possible. Whereas the MBCDI-2 percentiles were based on sex-adjusted norms, the other measures were not, but note that the two children with the overall highest performance were a girl and a boy. It is not possible to identify which components of the BBC had the greatest impact, if any, on speech, language, cognitive, and motor development. Many other variables in the study, for instance volubility of child vocalizations and environmental factors such as quantity of child-directed speech, remain to be analyzed, not only in this pilot cohort but also in the full set of families in this study. Longer-term outcomes in the primary and secondary outcome variables will be evaluated as we follow the children until age 4 years and evaluate a more complete spectrum of outcome variables including speech, language, cognitive and motor development, and quality of life. Most importantly, the trends toward beneficial effects of the BBC on the primary outcome variables of speech sound production and expressive language warrant appropriately powered larger clinical trials.\n\n\nData availability\n\nOpen Science Framework: Babble Boot Camp. https://doi.org/10.17605/OSF.IO/YZHT4 (Peter, 2019)\n\nThe project contains the following underlying data files:\n\n- UnderlyingData_MBL_SSL_MBCDI2.xlsx. This spreadsheet contains the MBL, SSL, and MBCDI-2 data for the participants.\n\nThe underlying data also include audio files recorded in the participants’ homes and hand-written questionnaires with identifiable information; therefore, these cannot be openly shared. De-identified questionnaire files can be made available to qualified researchers by contacting the first author at Beate.Peter@asu.edu.\n\nOpen Science Framework: Babble Boot Camp. https://doi.org/10.17605/OSF.IO/YZHT4 (Peter, 2019)\n\nThe project contains the following extended data files:\n\n- Extended Data 1: Flow diagram\n\n- Extended Data 2_Appendix_BBC_Program_v2\n\nOpen Science Framework: Babble Boot Camp. https://doi.org/10.17605/OSF.IO/YZHT4 (Peter, 2019)\n\nThe project contains the following reporting guidelines:\n\n- Reporting Guidelines 1: CONSORT checklist with Information to include in the abstract when reporting a pilot or feasibility trial\n\n- Reporting Guidelines 2: CONSORT checklist with Information to include in the manuscript when reporting a pilot or feasibility trial\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Grant information\n\nThis work was supported by a seed grand from the Arizona State University Institute for Social Science Research and an Arizona State University New Faculty Startup Fund award to B. Peter and by National Science Foundation (Resource Implementations for Data Intensive Research in the Social, Behavioral and Economic Sciences; 1539129) and the Washington Research Foundation to M. VanDam.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nMany thanks to the participating families for their time, effort, and faithful implementation of the program. We gratefully acknowledge Lauren Levanovic and Emilie Bonkrud for assistance with phonetic transcriptions, and Emma Williams and Caitlin Miner for assistance with administration of the program.\n\n\nReferences\n\nAmerican Speech-Language-Hearing Association: Childhood Apraxia of Speech [Position Statement]. 2007. Publisher Full Text\n\nAntshel KM, Epstein IO, Waisbren SE: Cognitive strengths and weaknesses in children and adolescents homozygous for the galactosemia Q188R mutation: a descriptive study. 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PubMed Abstract \| Publisher Full Text\n\nPaul R, Jennings P: Phonological behavior in toddlers with slow expressive language development. J Speech Hear Res. 1992; 35(1): 99–107. PubMed Abstract \| Publisher Full Text\n\nPaul R, Norbury CF: Language disorders from infancy through adolescence. Elsevier. 2012.\n\nPeter B: Babble Boot Camp. OSF. 2019. http://www.doi.org/10.17605/OSF.IO/YZHT4\n\nPotter NL, Nievergelt Y, Shriberg LD: Motor and speech disorders in classic galactosemia. JIMD Rep. 2013; 11: 31–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRogers SJ, Vismara L, Wagner AL, et al.: Autism treatment in the first year of life: a pilot study of infant start, a parent-implemented intervention for symptomatic infants. J Autism Dev Disord. 2014; 44(12): 2981–2995. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRowe ML, Goldin-Meadow S: Differences in early gesture explain SES disparities in child vocabulary size at school entry. Science. 2009; 323(5916): 951–953. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchonhaut L, Armijo I, Schönstedt M, et al.: Validity of the ages and stages questionnaires in term and preterm infants. Pediatrics. 2013; 131(5): e1468–1474. PubMed Abstract \| Publisher Full Text\n\nSemel E, Wiig E, Secord W: Clinical Evaluation of Language Fundamentals - Preschool - 2. Pearson. 2004. Reference Source\n\nShriberg LD, Tomblin JB, McSweeny JL: Prevalence of speech delay in 6-year-old children and comorbidity with language impairment. J Speech Lang Hear Res. 1999; 42(6): 1461–1481. PubMed Abstract \| Publisher Full Text\n\nSquires J, Bricker D: Ages & Stages Questionnaires. 3rd Ed. Brookes. 2009. Reference Source\n\nStoel-Gammon C: Constraints on consonant-vowel sequences in early words. J Child Lang. 1983; 10(2): 455–457. PubMed Abstract \| Publisher Full Text\n\nStoel-Gammon C: Prespeech and early speech development of two late talkers. First Lang. 1989; 9: 207–224. Publisher Full Text\n\nStoel-Gammon C: Relationships between lexical and phonological development in young children. J Child Lang. 2011; 38(1): 1–34. PubMed Abstract \| Publisher Full Text\n\nStorkel HL, Komesidou R, Fleming KK, et al.: Interactive Book Reading to Accelerate Word Learning by Kindergarten Children With Specific Language Impairment: Identifying Adequate Progress and Successful Learning Patterns. Lang Speech Hear Serv Sch. 2017; 48(2): 108–124. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTomblin JB, Records NL, Buckwalter P, et al.: Prevalence of specific language impairment in kindergarten children. J Speech Hear Res 1997; 40(6): 1245–1260. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVihman MM, Greenlee M: Individual differences in phonological development: ages one and three years. J Speech Hear Res. 1987; 30(4): 503–521. PubMed Abstract \| Publisher Full Text\n\nVihman MM, Macken MA, Miller R, et al.: From Babbling to Speech - a Re-Assessment of the Continuity Issue. Language. 1985; 61(2): 397–445. Publisher Full Text\n\nVygotsky LS: The Development of Higher Forms of Attention in Childhood. Sov Psychol. 1979; 18(1): 67–115. Publisher Full Text\n\nWaggoner DD, Buist NR, Donnell GN: Long-term prognosis in galactosaemia: results of a survey of 350 cases. J Inherit Metab Dis. 1990; 13(6): 802–818. PubMed Abstract \| Publisher Full Text" }	[ { "id": "46124", "date": "01 May 2019", "name": "Shelley Velleman", "expertise": [ "Reviewer Expertise Infant", "toddler", "and child speech development and disorders", "including prelinguistic vocalizations and early words", "and motor speech development and disorders." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript focuses on an important and relevant issue: determining whether proactive intervention strategies are beneficial for infants at risk for later speech or language deficits associated with a specific condition (Classic Galactosemia, henceforth “CG”). This is an ambitious longitudinal project. In this manuscript, the authors report on preliminary findings and directions for research.\n\nIs the work clearly and accurately presented and does it cite the current literature? The primary limitation of the work in its current form is a lack of specificity. First, the literature review fails to give the reader a full sense of the related research or of the related clinical services that have gone before. For example, the authors state that, “very early speech and language services are not yet available” (p. 3). They add that, “The question is whether proactive, preventive treatment, if it existed, could reduce the deleterious effects associated with the disease [that results in risk for speech-language delay] …and thereby improve outcomes” (p. 3). These statements seriously downplay decades of research and of speech-language services in early intervention. As long ago as 1983, Tomasello and Todd1 showed that levels of parental joint attention impacted their infants’ vocabularies between 12-18 months. Roberts and Kaiser’s systematic review2 documents the research demonstrating that training parents to interact with their children in certain ways can impact linguistic outcomes. For example, more recently, Roberts et al.3 trained caregivers to use enhanced milieu teaching using a “teach-model-coach-review” approach. This included not only telling and showing the caregivers what to do, but also coaching them as they tried out the strategies and reviewing their success afterwards. Treatment fidelity on the part of the trainers and on the part of the caregivers were both measured. These authors demonstrated that the frequency and accuracy with which the caregivers carried out the strategies that they were taught had an impact on the children’s communication progress. These are but a few of many studies documenting the value of SLPs training parents to interact with their infants and toddlers in a manner that will facilitate communication development, with the result that SLPs involved in early intervention typically do train parents to use these strategies. For example, ASHA’s4 practice portal document on early intervention states that, “Services that include opportunities for families and caregivers to directly participate in intervention are essential to strengthen existing knowledge and skills and to promote the development of new abilities that enhance child and family outcomes”5 and that, “SLPs often coach families, caregivers, and other team members in how to implement functional, language-enhancing strategies during daily activities (Brown, 20166; Brown & Woods, 20157, 20168; Roberts & Kaiser, 20112; Roberts, Kaiser, Wolfe, Bryant, & Spidalieri, 20143; Searcy, 20119; Shelden & Rush, 201010).”\n\nTherefore, the authors need to specify how their study is similar to and different from what’s been done before. Unique and important characteristics of their study might include the focus on stimulating prelinguistic vocalizations specifically (versus communication/cognition more generally), beginning at such a young age, and intervention for children with this specific syndrome. However, the very brief descriptions that they provide of their caregiver trainings make it impossible to judge exactly what they are training the parents to do, how they are training them to do it, and whether or not the treatment fidelity of the trainers or of the parents are actually measured. In this sense, the work is not clearly presented. See below for further discussion of the current lack of specificity in these areas.\n\nIs the study design appropriate and is the work technically sound? The authors are gathering specific data systematically from several children, selected according to specific criteria, including two control participants who are not receiving treatment (one of whom has CG and one of whom does not), across time. However, the authors need to explicitly state the study’s design at the beginning of methods section of this manuscript. Further, the work and its context within its content area and the methods used are not presented specifically enough to judge the soundness of the approach or the appropriateness of the conclusions that are drawn from the results.\n\nAre sufficient details of methods and analysis provided to allow replication by others? The authors do not provide adequate details about the participants and methods used in this study, specifically:\nInclusion and exclusion criteria should be organized separately on page 4 for clarity. In addition to participant criteria, authors should report participant characteristics (i.e., hearing/vision status, whether they are receiving other interventions concurrently, family composition/status, whether participants stay at home or are enrolled in daycare, etc.). This information is important to include for later analyses, as these variables may be confounders, mediators and moderators. In addition to participant information, characteristics need to be collected and provided about legal guardian(s), specifically (a) socioeconomic status, (b) level of education, and (c) estimated amount of time spent with participants (an important consideration for intervention implementation/ feasibility). The authors mention on page 4 that enrichment variables are monitored, but preliminary findings of how these variables may be influencing outcomes is important to report, even qualitatively. Again, this information is important to include for later analyses, as these variables may be confounders, mediators and moderators. Frequency of parent training and consultation is reported as “once per week for approximately 10 minutes” (p. 4). There is no information on the fidelity of parent implementation of BBC strategies, nor is there information on what types of training are provided. For example, were parents just given information or did trainers observe them using strategies and offer feedback? Roberts et al.3 recommend a four-step caregiver training process; Akamoglu and Dinnebeil11 recommend six steps. Were recommendations such as these followed? Were the parents’ goals for their children taken into account? Brown6 reports that, “Intervention studies using...a transactional [as opposed to uni-directional] coaching approach have shown to have positive effects on parent and child outcomes while aligning with the intent of family capacity-building (Brown & Woods, 20157, 20168; Roberts et al., 20143; Wetherby et al., 201412)” (p. 144). If fidelity measures were performed, how was it measured and what are the results? This is important for replication purposes and also for judging factors that may impact the results of the study. Roberts et al.3 report that, despite the intensive attention that was paid in their study to telling, showing, coaching, and reviewing the intervention strategies that they taught to parents, “generalization of strategy use to the home varied by caregiver and by strategy” (p. 1864) and also by activity type (play, book-reading, etc.). The fact that only approximately 10 minutes of training was provided weekly is problematic for clinical translation purposes, as this amount and frequency of SLP intervention is not billable or practical. The authors report that 50 utterances are compiled and transcribed using the International Phonetic Alphabet; however, no information is provided about how this information is acquired or under what circumstances. What level of IPA transcription is used - broad or narrow? Is IPA used for precanonical babble (which is not appropriate)? If not, are precanonical utterances omitted from the calculations? Are parents instructed to interact with their children during the samples using instructed strategies or with specific toys/stimuli? Are other communication partners involved, such as siblings? The authors deemed the participant pool insufficient for running statistical tests; however, more information needs to be provided qualitatively about the participants described in this pilot study. In terms of “quality control” on page 5, the authors say that scores were double-scored. The authors need to clarify whether they mean intra-rater or inter-rater reliability was performed. Rationale should also be provided as to why 15% of the data was selected for this type of reliability analysis.\n\nAre all the source data underlying the results available to ensure full reproducibility?\nThe use of symbols to mark data across time points in Figures 1-4 is problematic, as data points overlap in scores. This makes it difficult to determine whether two participants had the same score at one point or whether scores were not determined at particular time points. The authors should modify these figures using lines to present trends instead. If data are missing, then this needs to be indicated/reported, similar to what is reported if a participant is enrolled after 12 months of age. Since the primary outcome variables are speech sound production in babble, it would be advantageous for the authors to report information on the types and percentages of consonants and vowels that are acquired by the participants based on each 50 utterance sample compiled across time points. The same is also true about expressive language, specifically total number of words, word types, and stage 1 word types (i.e., operations of reference, semantic relations). These measures are valuable in monitoring progress and change over time, and are relevant based on the primary outcome variables of interest.\n\nAre the conclusions drawn adequately supported by the results?\nThe authors draw only tentative conclusions regarding why the children in the study had various outcomes, such as, “Whether these patterns reflect global levels of disease severity, global deficits from the treatment, or a combination of these cannot be ascertained based on the present data” (p. 8). This is appropriate, given the currently small sample size. However, as noted above, the authors should provide much more information about other factors (i.e., parental and environmental characteristics) that are likely to have influenced the outcomes of the children reported in this preliminary account. The authors state on page 5 that “Continued data collection and analysis in this population will provide conclusive evidence.” Given that the data reported are preliminary, this comment is appropriate. However, future evidence still should not be deemed as “conclusive,” but rather as “more promising”. It is too soon for the authors to draw conclusions about the validity of their measures based on these preliminary data. Therefore, their statement on page 7 that: “It is possible that MBL and SSL do not measure crucial weaknesses in children with CG in general” seems inappropriate at this time. The authors should omit or reword the phrase “Given the 90% risk for language impairment in children with CG” on page 8. This is a misinterpretation of the research reported by the authors on page 3: “language impairment in 90% of children with CG.” Risk should be reported as a statistical measure and cannot be inferred based on this reported proportion.\nWe look forward to further information and updates on this interesting study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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? Partly", "responses": [] }, { "id": "47831", "date": "31 May 2019", "name": "Barbara L. Davis", "expertise": [ "Reviewer Expertise Early periods of child speech acquisition in typically developing and at-risk populations." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary: This study reports on results of parent training for facilitating speech and language development for four young children with galactosemia and one control child whose parents were not trained in stimulation techniques. Outcome measures were compared with \"typical control children\" but the origin of that data was not described.\n\nIs the work clearly and accurately presented and does it cite the current literature? Citation about speech sound development is based on group studies of chronologically older children. No citations about variability in the pace of development. Would have been appropriate as the outcomes for these children are not supportive of the study rationale and could be due to variability either in diverse implementation by parents of the activities taught via telepractice, or diverse types of potential individual differences in the children. No literature review of parent training techniques, the major implementation method used. Child outcome measures carefully described. No description of outcomes of parent training provided. Those might help to understand data outcomes on child variables measured.\n\nIs the study design appropriate and is the work technically sound? In this developmental period, parent training is a well established way to conduct intervention. Some outcome variables focused on intervention fidelity and range of potential differences across parents would be appropriate .\n\nAre sufficient details of methods and analysis provided to allow replication by others? Very good information on child measurements with respected analyses or tests. No information about parent input analyses. I do not believe they are available.\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable for this pilot study.\nAre all the source data underlying the results available to ensure full reproducibility? Raw data not available but types of analyses are standards in the field. This aspect of the study is well described.\nAre the conclusions drawn adequately supported by the results? In this pilot cohort of four CG children, one control, and and data from a group of comparison TD children, results do not support assertions made in Discussion. No measure of parent behaviors, the method of implementation for BBC intervention method. Either diverse implementation by parents of the activities they were taught via telepractice, or diverse types of potential individual differences in the children could account for the findings in the pilot data on MBL and SSL the primary issue of interest in measuring BBC intervention outcomes. I am very supportive of the importance of intervention research and focus on early intervention in ‘at risk’ populations. Unfortunately, the number of unknowns that are not yet accounted for in this pilot study of four children and a control suggests that it may be a premature to make assertions about effects of BBC parent intervention with CG children based on this dataset.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/8-271
https://f1000research.com/articles/9-425/v1	22 May 20	{ "type": "Study Protocol", "title": "The effects of a temporal processing-based auditory training program on the auditory skills of elderly users of hearing aids: a study protocol for a randomized clinical trial", "authors": [ "Nariman Rahbar", "Karim Sattari", "Mohsen Ahadi", "Hamid Haghani", "Nariman Rahbar", "Mohsen Ahadi", "Hamid Haghani" ], "abstract": "Background: One of the most important effects of age-related declines in neural processing speed is the impairment of temporal resolution, which leads to difficulty hearing in noisy environments. Since the central auditory system is highly plastic, by designing and implementing a temporal processing-based auditory training program, we can help the elderly improve their listening skills and speech understanding in noisy environments. Methods: In the first phase of this research, based on the theoretical framework of temporal processing, an auditory training solution was developed as a software program. In the second phase, which will be described in the present study, the effects of the designed program on the listening skills of the elderly users of hearing aids (age: 60-75 years) will be studied in the control and intervention groups. In the intervention group, the auditory training program will be implemented for three months (36 sessions), and the results of central tests (GIN, DPT, QuickSIN) and the electrophysiological speech-ABR test will be compared in both groups before, immediately and one month after the intervention. Discussion: Since temporal processing is not sufficient in auditory training programs for the elderly with hearing impairments, implementation of a temporal processing-based auditory training program can reduce hearing problems in noisy environments among elderly users of hearing aids. Trial registration: This study was registered as a clinical trial in the Iranian Registry of Clinical Trials (IRCT20190921044838N1) on December 25, 2019.", "keywords": [ "Auditory training", "Temporal processing", "Hearing aid", "Age-related Hearing loss" ], "content": "Introduction\n\nOne of the most important issues in today’s world is the phenomenon of aging. With advancing age, the prevalence of chronic diseases increases. Statistics show that at least one chronic disease occurs in 80% of the elderly over 65 years1. According to previous findings, the prevalence of chronic diseases increases up to the age of 75 years. One of the most common problems facing the elderly is hearing loss. Hearing loss is the second most common health problem following arthritis in the elderly over 65 years1.\n\nAge-related hearing loss or presbycusis is a progressive disease, characterized by audiometric threshold shift and impaired speech comprehension, especially in noisy environments2,3. Many studies have shown reduced speech comprehension in the presence of competitive noise in the elderly, even with normal hearing sensitivity4–8. Difficulty in speech comprehension in noisy environments has been attributed to a variety of factors, which are not fully understood4. Nonetheless, evidence shows that defects in the peripheral auditory system, retrocochlear processing, and central auditory processing may contribute7.\n\nOne of the important factors in speech comprehension in noisy environments is precise neural timing. With advancing age, individuals need more time to respond to sensory input and develop neural slowing. One of the most pronounced outcomes of neural slowing is damage to temporal resolution, which leads to difficulty hearing in noisy environments9. There are many potential biological reasons for the elderly’s slower responses, such as reduced myelin integrity, prolonged nerve recovery, reduced brain connections, and reduced neural synchrony. Perceptual and neurophysiological studies have shown that central auditory processing is also impaired by hearing loss and aging. Among central auditory processing skills, those related to temporal processing are more influenced by age than others10.\n\nMany studies have shown the effects of auditory training on cortical plasticity in animals11–13 and humans14,15. Generally, neuroplasticity is not specific to the cortex, which receives input from the brainstem and thalamus16, and some animal studies have indicated plasticity in subcortical structures17–22. However, there are limited studies on subcortical plasticity among the elderly following auditory training and education23. Music therapy has been used to improve speech perception in the presence of noise in the elderly, and it is believed to be effective in improving the separation of simultaneous speech sounds by enhancing basic frequency and harmonic reception24–27.\n\nWe can modify the physiological representation of sounds using training exercises, as training-dependent physiological changes can lead to improved perception; this phenomenon is sometimes referred to as learning-related plasticity28. In animal studies, it was found that with training, we can change auditory mapping and time decoding. Overall, training-related physiological changes can result in several processes, including an increased number of neurons responsive to the sensory field, increased neuronal synchrony, and neuronal de-correlative processes, associated with inconsistent neuron activity (i.e., each neuron has a specific and distinct function). In other words, with training, neurons in the same category are arranged in a way that they can represent all of the distinct and minor features of the stimulus. In this process, it is assumed that information shared between two stimuli is ignored, but the response to the distinct features of each stimulus is increased28.\n\nToday, modulation of the auditory system using training exercises to enhance perception is one of the most important research trends. Substantial evidence suggests that humans, with or without hearing loss, can increase their ability to perceive spectral and temporal features of acoustic stimuli through auditory training exercises. Auditory training exercises, which are mainly classified into analytical (bottom-up), combined (top-down), and mixed (combination of bottom-up and top-down methods) types, are designed to improve the individual’s ability to understand auditory events through repeated listening exercises29.\n\nAnalytical exercises emphasize the acoustic content of the signal (i.e., spectral, temporal, and intensity features), and one's task is to identify and differentiate different sounds. On the other hand, combined training seeks to improve one’s signal understanding by enhancing attention to stimuli, combining the stimuli, and using contextual information29. Today, most auditory training programs, designed for individuals with communication disorders, use analytical models, which are also considered in the present study. In this study, we aimed to determine the relationship between the temporal characteristics of speech processing, as the basis of speech perception in noisy environments with competitive sounds, and temporal processing-based auditory training.\n\n\nMethods\n\nThe used tests do not cause any complications for the participants, and the participants can withdraw from the study at any stage. All auditory training examinations and sessions are free of charge. Participants with knowledge of objects and details of the research will be included after their written informed consent is obtained by one of the researchers of this study. The nature of the tests and rehabilitation program is such that there are no side effects or harm for participants. The consent form is provided as Extended data30. The confidentiality of data will be respected during the study as explained in the consent form. If the results are positive, the auditory training program will be also implemented for the control group. All ethical principles of the Medical Ethics Committee of IUMS will be respected in this study and two anonymous reviewers will be chosen by the deputy of the IUMS research review board to assess the final report of the study. The study does not have an audit and only the final report of the research project will be submitted. The Medical Ethics Committee approved the study protocol (IR.IUMS.REC.1397.652). Researchers will send any amendments to the protocol in the future to the ethics committee.\n\nAll data will be entered into forms which are prepared for data collection (see Table 3; Extended data)30 and the participant files will be stored at study site and will be maintained in a secure place and manner. Participants' files will be stored securely after completion of the study and the study data will be available publicly on a repository.\n\nThis clinical trial protocol is the second part of a larger research project. In the first part, the intervention was developed based on the theoretical framework of temporal processing, which is available as an auditory-based training software program (at home PC-based training program)31. In the second part (simple randomized clinical trial), the effect of the designed training program will be examined. For this purpose, authors will evaluate 60–75 years-old elderly with hearing loss, who live in Tehran and are referred to the Audiology Clinic of the Rehabilitation School of Iran University of Medical Sciences with more than three months of experience in using binaural hearing aids and will be selected based on the inclusion criteria for entering the study. Participants will be invited to take part in the study face-to-face at the clinic.\n\nThe participants will be randomly divided into two groups. The intervention group will participate in the auditory training program, while the control group will not be involved in the auditory training program; both groups will be matched for age and gender. For this purpose, random numbers are assigned to individuals based on a random number table. Subjects with odd numbers are assigned to the control group, while those with even numbers are assigned to the intervention group. This process will be carried out by an audiology department employee, who is not involved in the study. Allocation concealment will be performed by random allocation cards using computer-generated random numbers.\n\nThe inclusion criteria are as follows: 1) age range of 60–75 years; 2) mild to moderate bilateral sensorineural hearing loss; 3) mean hearing threshold of 40–55 dBHL (averaged over 0.5, 1, 2, and 4 kHz) in both ears; 4) bilateral hearing loss; 5) use of binaural hearing aids for more than three months; 6) normal middle ear function32; 7) lack of cognitive problems based on the results of Mini-Mental State Examination (MMSE)33; 8) having a high-school diploma or higher; 9) right-handedness based on the Edinburgh Handedness Inventory34; and 10) being a monolingual (Farsi).\n\nUnwillingness to cooperate with the study at any stage and failure to meet any of the inclusion criteria.\n\nThis research consists of two phases, the first of which has been already carried out.\n\nPhase 1: Design and development of the temporal processing-based auditory training program. The auditory training program was designed based on the theoretical framework of temporal processing and was presented to 10 academic experts to determine its content validity. After that, the program was reviewed by an expert panel, and disagreements were resolved. The experts were academic members of IUMS, Tehran University of Medical Sciences (TUMS), and Shahid Beheshti University of Medical Sciences (SBUMS). This program has been approved by the Medical Ethics Committee of Iran University of Medical Sciences (IR.IUMS.REC.1397.652) and is described in detail below.\n\nThe program is based on different aspects of temporal processing consisting of five tasks while one or more aspects are challenged in all tasks, including:\n\n1. Detecting the number of stimuli\n\nThis item contains five stimuli (frequencies of 500, 1000, 2000, 4000 Hz and white noise) that are presented in single, binary, or ternary with different combinations for the users and their task is to count the number of stimuli heard. In the first level session, the duration of the stimuli and the gap between the stimuli is 250 ms. In each session, the duration and gap between stimuli is decreased by 5 ms, so in the last session (session 36) the duration and the gap between the stimuli reaches 75 ms.\n\n2. Detecting the pitch of the stimuli\n\nThis item contains five stimuli (frequencies of 250, 500, 1000, 2000, 4000 Hz) that are presented in single, binary, or ternary with different combinations for the users and their task is to tell the number of stimuli that are similar in frequency. In the first session, duration of the stimuli and the gap between the stimuli is 1000 ms. In each session, the duration and gap between stimuli decreases by 25 ms, so in the last session (session 36) the duration and the gap between the stimuli reaches 125 ms. The initial sessions use the stimuli that have the highest octave distance (four octaves), while in the final sessions the distance is minimized (one octave).\n\n3. Detecting the duration pattern\n\nThis item contains three stimuli (frequencies of 500, 1000, 2000 Hz) and each stimulus is provided to the users with different triple combinations of long or short duration and their task is to determine the number of stimuli that are similar in terms of duration. In the easy stage (1st to 4th week) the long duration is 600 ms and the short duration is 300 ms. However, the gap between the stimuli in the first week is 250 ms, which decreases by 50 ms per week and reduces to 100 ms in the fourth week. The stimulus of the first session of every week is 500 Hz, the second session is 1000 Hz and the third session is 2000 Hz. The same is true for the medium stage (fifth to the eighth week) and the hard stage (9th to 12th week). In the medium stage, the long stimulus duration is 500 ms and the short stimulus duration is 250 ms and at the hard stage the long stimulus duration is 400 ms and the short stimulus duration is 200 ms.\n\n4. Detecting the number of nonsense speech stimuli in noise\n\nThis item contains three nonsense speech stimuli (da / ta / ba) with different signal-to-noise ratios for both male and female voices in single, binary or ternary and in different combinations are provided in the presence of background noise and the task is to tell the number of stimuli they have heard. The signal-to-noise ratio starts at 12 dB in the first session, and both sessions decrease by 1 dB (one session for male and one for female), until finally, in the last two sessions, the signal-to-noise ratio decreases to -5 dB.\n\n5. Detecting the gap in noise\n\nThe stimulus of this item is white noise, which is provided to clients with a 6-second duration, with zero to three gaps of different durations, and they should determine the number of gaps they have heard. The gap duration starts from 40 - 41 - 42 ms in the first session and each session decreases by 1 ms until session 33, when each session decreases by 0.5 ms until the last session (36) when it has decreased to 7.5 - 8 - 8.5 ms (see Table 1, Extended data)30.\n\nThe program consists of three stages (easy, medium, and hard), each with 12 levels (sessions). At each level, five temporal processing items are considered with five exercises. Since each exercise should be repeated twice, each auditory training session consists of 50 exercises, which are performed by the subject according to the description of each item. Since the program consists of 36 sessions, the auditory training program comprises 1800 exercises (see Table 2, Extended data)30. One or more aspects of temporal processing are challenged in all items. In the first sessions, the user’s task is simple, while in the subsequent sessions, it becomes more difficult, requiring greater precision. Indeed, the program is based on the repetition of exercises.\n\nPhase 2: Effect of the temporal processing-based auditory training program. This phase will be performed in three steps: before, during, and after the training program.\n\n1. Assessments before the auditory training program:\n\nIn this phase, the following assessments will be performed by researchers for both the control and intervention groups in the audiology clinic of IUMS:\n\nThe subject’s case history will be taken to examine the inclusion and exclusion criteria.\n\nOtoscopy and behavioral tests, including pure-tone average (PTA), speech tests such as speech recognition threshold (SRT) and speech discrimination score (SDS), and tympanometry, will be performed.\n\nMMSE will be applied to assess the absence of cognitive impairment in the participants.\n\nAn electrophysiological test of speech auditory brainstem response (ABR) will be carried out. By studying and comparing the changes in amplitude, latency, and content of frequency responses, the possible effects of the auditory training intervention on the isolation of simultaneous speech sounds will be determined electrophysiologically. Regarding the objective nature of changes in electrophysiological tests, electrophysiological evidence can confirm and demonstrate behavioral changes. Since speech ABR is a suitable test for evaluating subcortical auditory processing mechanisms in speech comprehension in the presence of background noise35, a clear relationship between the stimuli and brainstem responses allows for direct comparisons between frequency and temporal components of the stimulus and response. Previous research has shown a relationship between speech comprehension in the presence of background noise and spectral and temporal components of speech ABR in children4, adults36, and the elderly4. Changes in this test may be an objective reason for altering the function of the nervous system.\n\nAn ABR test will be performed with a speech stimulus, using the Bio-Logic Navigator Pro System. In this test, a non-inverting electrode is placed at Cz, and the inverting electrode is placed on the right ear lobule, and a ground electrode is placed on the forehead. The impedance value of all electrodes is below 5 kΩ (maximum=1.5 kΩ). The stimulus is a 40-msec synthetic /da/ syllable, used in previous studies by the Auditory Neuroscience Laboratory of Nina Kraus et al.15 at Northwestern University (USA). The test will be performed in a quiet place with closed eyes (or during sleep) while leaning against a comfortable chair in an acoustic room with low light and low ambient noise. No cognitive stimulus will be used during the experiments7.\n\nCentral tests, including gap detection in noise (GIN), duration pattern test (DPT), and QuickSIN, will be performed. These tests are sensitive to the central auditory nervous system (CANS) lesions but are not affected by peripheral hearing loss37.\n\nGIN test: This test consists of a series of 36 different six-second segments of white noise. Each segment contains zero to three gaps of silence. The interval between the noise segments is five seconds, and the duration of silence gaps is 2, 3, 4, 5, 6, 8, 10, 12, 15, and 20 msec. Ten practice items are presented before starting the test. There are four lists available for the test, which allow for a comparison of the tests. Generally, one list is used for each ear. Although the test is run through a single channel, a second channel is also used by the examiner to control and rate the responses. The test includes two criteria for examining the results: 1) a threshold for detecting gaps in noise; and 2) the percentage of correct responses for each ear. The gap detection threshold in noise is considered as the shortest gap duration for which there are four out of six correct responses. The percentage of correct responses is the mean percentage of correct responses during the test38.\n\nDPT test: This test consists of three pure tones in each segment. The frequency of each tone is 1000 Hz, and its duration is either 250 msec (short) or 500 msec (long). The gap between the tones is 300 msec. Generally, there are six patterns for this test (LLS, LSL, LSS, SLS, SLS, SLL, and SSL), and a six-second gap of silence separates the segments. The percentage of correct responses is the mean of all percentages of one's correct responses during the test. It is recommended to perform the test at 50 dB SL to the speech recognition threshold or pure tone average (500, 1000, and 2000 Hz). Since intensity has insignificant effects on test performance, it can be also performed at 10 dBSL38.\n\nQuickSIN test: In general, QuickSIN is a quick test to quantify one’s ability to hear in the presence of noise. It can be performed using headphones and/or free-field speakers (free-field in this study). In this test, a series of sentences are presented to both ears, and at the same time, a babbling noise is presented to both ears; in other words, noise is competitive with the stimulus. This test is performed at the most comfortable level (MCL). The patient's response is written, and the number of correct repeated words is documented in the “results” sheet. A total of five lists are used for each person.\n\nEach list contains six sentences, and each sentence has five keywords, all of which are presented in babble noise. The noise level is set at six levels (preset on CD) and increases in 5-dB steps; in other words, the test conditions gradually become more and more competitive. The signal-to-noise ratios include 25, 20, 15, 10, 5, and 0 dB. At the end of each list, the total number of correct repeated words is recorded, and the signal-to-noise reduction ratio (SNR loss) is calculated32.\n\n2. Implementation of the temporal processing-based auditory training program (only for the intervention group)\n\nMultiple studies have shown that plasticity occurs in the brain 8–12 weeks after training39. Therefore, after the study population is selected, the designed auditory training program will run only for the intervention group over three months. The program will be provided as a DVD to the intervention group for use at home (at home PC-based training program). The auditory training program will be carried out in three easy, medium, and hard stages, with each stage consisting of 12 levels (sessions). Each stage will continue for one month and will be performed by the user for three sessions per week (30–40 minutes per session).\n\nIn the designed program, the difficulty of training increases from level 1 to level 36, and the user’s task becomes more difficult requiring more precision. The program is based on the repetition of exercises. In each session, the user is required to earn more than 80% of the scores to pass to the next level. All of the exercises will be performed in several stages over a three-month auditory training period. The exercises start from easy levels in the early days to difficult levels in the final weeks. The program will be implemented at MCL for users with hearing aids in the free field.\n\nTo fully familiarize the user with the auditory training program, the first three sessions of the program will be conducted in the audiology clinic in the presence of the researcher to ensure that the user has learned how to use the program correctly and that it is executed properly. Subsequent home sessions will be conducted according to the program protocol and the training of each part of the program. However, in order to retain control and ensure that the program is performed correctly, in all rehabilitation sessions at home, users will be monitored by the researcher via a phone or video call. If a participant is unable to run the program after these tutorials and tips, the training will be discontinued and the person will be removed from the study.\n\n3. Assessments after the implementation of the auditory training program\n\nIn this stage, central tests, including GIN, DPT, QuickSIN, and speech ABR test, will be repeated for both the control and intervention groups immediately after auditory training, and the outcomes will be compared with the pre-training results. The tests will be repeated after one month to determine the reliability of intervention results to improve the users’ temporal processing.\n\nThe sample size is calculated with 80% power and 5% test error (95% confidence interval), based on a similar study by Gil et al.39:\n\n\n\nAccording to the abovementioned formula and a 20% dropout rate, the sample size is estimated to be 22 in this study (11 participants per group).\n\nFirst, the Chi-square test will be used to examine the normal distribution of each variable. If data distribution is normal, parametric tests, including paired t-test, independent t-test, and ANCOVA test, will be performed. Otherwise, non-parametric Mann-Whitney and Wilcoxon tests will be carried out. Fisher’s exact test will be applied for the homogeneity of the groups. Statistical analysis will be conducted using SPSS version 22.0 (IBM Corporation, New York, USA), and the significance level is considered to be 0.05.\n\nThis trial will start in April 2020 and will be completed in September 2020. To date, enrolment of patients has been performed, and allocation will be done shortly. The trial outcomes will be published in the relevant scientific journals and the results will be communicated to the public, participants and audiologists through a formal report. The results of this study will be communicated to the external funding body through a formal report and there is no limit on the publication of the trial results.\n\n\nDiscussion\n\nAge-related hearing loss not only influences the physical and emotional activities of the elderly, but also affects their social activities and reduces their quality of life due to various factors, such as depression, social isolation, and reduced self-confidence. One of the important problems in maintaining and promoting the health and quality of life of the elderly is to maintain their independence in daily life activities and to provide suitable conditions for them to continue an active and independent life40,41. Therefore, by implementing a proper auditory training program, the elderly can comprehend speech in noisy environments; this, in turn, prevents their isolation and reduces the social and financial burdens of their disabilities.\n\nThe “plasticity” phenomenon, which has been scientifically approved, suggests the efficacy of auditory training in improving the speech comprehension skills of the elderly. So far, various auditory training methods have been introduced and applied to different studies42. In Iran, we need to design and develop such methods based on the elderly’s needs. Multiple studies have been performed on auditory training for children after receiving hearing aids. However, in Iran, auditory training for elderly users of hearing aids has not been studied, and this is the first research conducted in our country. For this purpose, an elderly auditory training program was designed and implemented with an emphasis on the temporal aspects of speech. Also, regarding the need for special tools and facilities to establish this auditory training program, the proposed program can be used in audiology clinics and academic centers to improve the speech comprehension of elderly users of hearing aids, especially in the presence of background noise.\n\n\nData availability\n\nNo underlying data are associated with this article.\n\nOpen Science Framework: The effects of a temporal processing-based auditory training program on the auditory skills of elderly users of hearing aids: a study protocol for a randomized clinical trial. https://doi.org/10.17605/OSF.IO/SB93430\n\nThis study contains the following extended data:\n\nConsent form for participants of the study.docx\n\nTable 1 (Details and steps of the rehabilitation program).xlsx\n\nTable 2 (Schedule of temporal processing-based program).docx\n\nTable 3 (The data collection sheet of the study).docx\n\nTable 4 (Participant timeline).docx\n\nOpen Science Framework: SPIRIT checklist for 'The effects of a temporal processing-based auditory training program on the auditory skills of elderly users of hearing aids: a study protocol for a randomized clinical trial': https://doi.org/10.17605/OSF.IO/QPJDK30.\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\nSource code available from: https://github.com/karimsattari/source-code-of-auditory-training/tree/V2.0.0\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.381673531\n\nLicense: MIT", "appendix": "References\n\nCalhoun K, Eibling D: Geriatric otolaryngology. 1st ed. New York: Taylor & Francis Group; 2006. Reference Source\n\nMerchant SN, Nadol JB: Schuknecht's pathology of the ear. 3rd ed. Shelton, CT : People's Medical Pub. House-USA; 2010. Reference Source\n\nMatthews LJ, Lee FS, Mills JH, et al.: Extended high-frequency thresholds in older adults. J Speech Lang Hear Res. 1997; 40(1): 208–14. PubMed Abstract \| Publisher Full Text\n\nAnderson S, Parbery-Clark A, Yi HG, et al.: A neural basis of speech-in-noise perception in older adults. Ear Hear. 2011; 32(6): 750–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDu Y, Kong L, Wang Q, et al.: Auditory frequency-following response: a neurophysiological measure for studying the \"cocktail-party problem\". Neurosci Biobehav Rev. 2011; 35(10): 2046–57. PubMed Abstract \| Publisher Full Text\n\nGetzmann S, Wascher E, Falkenstein M: What does successful speech-in-noise perception in aging depend on? Electrophysiological correlates of high and low performance in older adults. Neuropsychologia. 2015; 70: 43–57. PubMed Abstract \| Publisher Full Text\n\nVander Werff KR, Burns KS: Brain stem responses to speech in younger and older adults. Ear Hear. 2011; 32(2): 168–80. PubMed Abstract \| Publisher Full Text\n\nPichora-Fuller K, MacDonald E: Auditory temporal processing deficits in older listeners: From a review to a future view of Presbycusis. Proceedings of the International Symposium on Auditory and Audiological Research; 2007. Reference Source\n\nAnderson S, White-Schwoch T, Parbery-Clark A, et al.: Reversal of age-related neural timing delays with training. Proc Natl Acad Sci U S A. 2013; 110(11): 4357–62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFitzgibbons PJ, Gordon-Salant S: Age-related differences in discrimination of temporal intervals in accented tone sequences. Hear Res. 2010; 264(1–2): 41–7. 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PubMed Abstract \| Publisher Full Text\n\nRusso NM, Nicol TG, Zecker SG, et al.: Auditory training improves neural timing in the human brainstem. Behav Brain Res. 2005; 156(1): 95–103. PubMed Abstract \| Publisher Full Text\n\nEdeline JM, Weinberger NM: Subcortical adaptive filtering in the auditory system: Associative receptive field plasticity in the dorsal medial geniculate body. Behav Neurosci. 1991; 105(1): 154–75. PubMed Abstract \| Publisher Full Text\n\nEdeline JM, Weinberger NM: Thalamic short-term plasticity in the auditory system: associative returning of receptive fields in the ventral medial geniculate body. Behav Neurosci. 1991; 105(5): 618–39. PubMed Abstract \| Publisher Full Text\n\nEdeline JM, Weinberger NM: Associative retuning in the thalamic source of input to the amygdala and auditory cortex: receptive field plasticity in the medial division of the medial geniculate body. Behav Neurosci. 1992; 106(1): 81–105. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStrait DL, Slater J, O'Connell S, et al.: Music training relates to the development of neural mechanisms of selective auditory attention. Dev Cogn Neurosci. 2015; 12: 94–104. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwaminathan J, Mason CR, Streeter TM, et al.: Musical training, individual differences and the cocktail party problem. Sci Rep. 2015; 5: 11628. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTierney AT, Krizman J, Kraus N: Music training alters the course of adolescent auditory development. Proc Natl Acad Sci U S A. 2015; 112(32): 10062–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTremblay KL: Beyond the ear: Physiological perspectives on auditory rehabilitation. Semin Hear. 2005; 26(03): 127–36. Publisher Full Text\n\nAlain C, Snyder JS, He Y, et al.: Changes in auditory cortex parallel rapid perceptual learning. Cereb Cortex. 2007; 17(5): 1074–84. PubMed Abstract \| Publisher Full Text\n\nSattari K: The effects of a temporal processing-based auditory training program on the auditory skills of elderly users of hearing aids: a study protocol for a randomized clinical trial. 2020. http://www.doi.org/10.17605/OSF.IO/SB934\n\nkarimsattari: karimsattari/source-code-of-auditory-training: auditory training software (Version V2.0.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3816735\n\nKatz J: Handbook of clinical audiology. Baltimore: Medwestsky. Burkard & Hood; 2009. Reference Source\n\nForoughan M, Jafari Z, Shirin Bayan P, et al.: Validation of mini-mental state examination (MMSE) in the elderly population of Tehran. Adv Cognit Sci. 2008; 10(2): 29–37. Reference Source\n\nOldfield RC: The assessment and analysis of handedness: the Edinburgh inventory. Neuropsychologia. 1971; 9(1): 97–113. PubMed Abstract \| Publisher Full Text\n\nAnderson S, Kraus N: Objective neural indices of speech-in-noise perception. Trends Amplif. 2010; 14(2): 73–83. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSong JH, Skoe E, Banai K, et al.: Perception of speech in noise: neural correlates. J Cogn Neurosci. 2011; 23(9): 2268–79. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGordon-Salant S, Fitzgibbons PJ: Profile of auditory temporal processing in older listeners. J Speech Lang Hear Res. 1999; 42(2): 300–11. PubMed Abstract \| Publisher Full Text\n\nChermak GD, Musiek FE: Handbook of central auditory processing disorder. Cambridge Univ Press; 2003.\n\nGil D, Iorio MC: Formal auditory training in adult hearing aid users. Clinics (Sao Paulo). 2010; 65(2): 165–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArslantas D, Unsal A, Metintas S, et al.: Life quality and daily life activities of elderly people in rural areas, Eskisehir (Turkey). Arch Gerontol Geriatr. 2009; 48(2): 127–31. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "63907", "date": "01 Jun 2020", "name": "Ahmadreza Nazeri", "expertise": [ "Reviewer Expertise hearing science", "amplification", "Electrophysiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 design tries to introduce an innovative temporal processing training software, which will be able to improve temporal processing abilities in the elderly population.\nA complete test battery will be able to show the possible improvements of the above mentioned temporal processing training program in a hearing-impaired elderly population.\nI have a recommendation for you, it would be better to draw a flowchart and show all the details inside it; however, it is only a recommendation\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": [] }, { "id": "64063", "date": "15 Jun 2020", "name": "Maria Cecília Martinelli Iório", "expertise": [ "Reviewer Expertise Cognitive Hearing" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe abstract is adequately presented. The introduction justifies the completion of the study and the methods provided allow the replication by others. The study is important in the area of knowledge of audiology, especially because from this knowledge the indication of rehabilitation can be better conducted. Bibliographic references must be updated. I understand that the best reference to justify the prevalence of hearing loss in the elderly is the global burden of disease. Other references must be updated too. Vos, T., Abajobir, A. A., Abate, K. H., Abbafati, C., Abbas, K. M., Abd-Allah, F., Abera, S. F. (2017). Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016. The Lancet,390(10100),1211-1259.doi:https://doi.org/10.1016/S01406736(17)32154-21\n\nAs the text explains age-related hearing loss (ARHL) not only influences the physical and emotional activities of the elderly, but also affects their social activities and reduces their quality of life due to various factors, such as depression, social isolation, and reduced self-confidence. One of the important problems in maintaining and promoting the health and quality of life of the elderly is to maintain their independence in daily life activities and to provide suitable conditions for them to continue an active and independent life.\n\nI think that the application of questionnaires that evaluate the limitations and restrictions in the activities of daily life is of great importance. Tests in controlled situations that do not represent daily activities may not show the improvement targeted by the training.\n\nTherefore, I believe that it is very important to apply questionnaires that assess the quality of life and the limitations and restrictions in activities of daily living before and after auditory training.\n\nIf it is not possible to include this type of evaluation, consider these aspects when discussing the results and the possible applicability of the proposed training.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "63904", "date": "16 Jun 2020", "name": "Amineh Koravand", "expertise": [ "Reviewer Expertise Neuro-audiology", "speech perception", "central auditory processing", "enriched auditory experiences", "cortical and subcortical auditory evoked potentials." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is a well-written study protocol. The need to have an auditory training program designed for the elderly is critical and very important. The authors are addressing this need by developing a special auditory software. I approve the protocol; however, I do have some comments for improving the study. Most of them are minor and can be handled easily. My two main concerns (explained below under the method section) are related to the participants ‘age and adding a speech perception test at the beginning of the experiment (as a screening tool for measuring the participant's qualification).\n\nIntroduction:\n\nThe introduction is short, but it may be related to the Journal specification for the study protocol. Some concept needs to be explained briefly: neural slowing, plasticity, the theoretical framework of temporal processing’, speech perception difficulties, how were these difficulties were measured in the past Some references need to be added. The authors advised to re-read the introduction and add the appropriate references. I am reporting several here, however, the authors need to check the introduction again:\n\nFor example: The first paragraph of the introduction (the first 2 sentences): ‘One of the most important issues in today’s world is the phenomenon of aging’. ‘With advancing age, the prevalence of chronic diseases increases’. Third Paragraph: ‘There are many potential biological reasons for the elderly’s slower responses, such as reduced myelin integrity, prolonged nerve recovery, reduced brain connections, and reduced neural synchrony’ and the next sentence: ‘Perceptual and neurophysiological studies have shown that central auditory processing is also impaired by hearing loss and aging.’ Sex paragraph: 'Substantial evidence suggests that humans, with or without hearing loss, can increase their ability to perceive spectral and temporal features of acoustic stimuli through auditory training exercises.'\nObjective/Aim The last paragraph of the introduction is related to the objective. This needs to be re-written. The main objective of the project needs to be presented more clearly. Can the authors formulate one or two hypotheses for their project?\nMethod section: Inclusion criteria: Numbers 3 and 4 can be combined. Number 5: why 3 months, based on which studies. It will be highly necessary to include one speech perception test (in silence and in noise) for selecting the participant. In fact, not all the participants would have an important speech understanding difficulty in noise. Can the speech perception be measured before enrolling in the project? Since the authors will run a basic audiological evaluation to determine the hearing loss degree, adding a speech test (even a screening one), will be so beneficial.\n\nParticipant's age: I am a little concerned about the participant’s age range, it is an important factor. A 60 years old participant will not have the same auditory capacity (Peripheral and central) compared to a 75 yrs old. Do you have any distribution limits? I mean how would the authors control this potential situation: 10 out of 12 participants are 60 yrs and only 2 are 75 years in the experimental group. I understand that we cannot control all the variables, however, can we concentrate on 70 to 75 yrs population instead of having this large age window (especially for the elderly that each year would bring some kind of declines in their capacity)\n\nTesting the Auditory training’ effect: Before, immediately and one month after. Can we evaluate the performance three months later as well to be sure about the long-term effect?\nAuthors mentioned that the training sessions will be monitored via phone or video call, is it for each session? How the authors can be sure that the participants will be practicing 30 to 40 minutes per session (not more or less), is there any way to detect the duration of the practice.\nDiscussion: Authors mentioned that age-related hearing loss not only influences the physical and emotional activities of the elderly but also affects their social activities and reduces their quality of life. Is it possible to add a questionnaire evaluating some of the emotional, social, and/or daily communication patterns?\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "63905", "date": "30 Jun 2020", "name": "Helen Henshaw", "expertise": [ "Reviewer Expertise Adult aural rehabilitation", "auditory training", "cognitive training", "health behaviour change", "cognition", "coproduction" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe abstract discussion section is a little unclear and could be misinterpreted as it currently stands. Suggest rewording the sentence 'Since temporal processing is not sufficient in auditory training programs for the elderly with hearing impairments' for clarity.\nJustification should be offered for each of the inclusion criteria listed.\nJustification should be offered for the limited age range of eligible participants (60-75 years old).\nPlease state your criteria for pass/fail on the MMSE.\nThe authors should add more detail in terms of how trainees progress through the training difficulty levels on the training program. Given that the training is DVD-based, is an adaptive algorithm based on trainee performance possible? Please describe in detail how transitions between difficulty levels will be achieved and managed.\nSimilarly, will the training log user input and performance in real-time? On the local PC? How/will these data be accessed and used by the research team? Or is on-task training performance not gathered/assessed?\nIf participants are removed from the study, how will this be dealt with (i.e. will they be replaced?)\nDo the authors intend to conduct per-protocol or intention-to-treat analyses? The data analysis section is limited and would benefit from the consideration of multivariate techniques aligned to this type of research (multiple outcome assessments over time).\nThe authors should state the outcome data on which the power calculation is based, particularly given that the proposed group size is very small in comparison to other computer-based auditory training intervention studies (see e.g. Henshaw & Ferguson, 2013 for a review)1. Notably, the sample size in Gil (which the authors use as the basis for their calculation) was 'doubled' by including participant data for right and left ears independently (not recommended). I would therefore strongly urge the authors to revisit their power calculation based on pilot data and/or a high quality published study using aligned outcome measures in a similar population.\nI commend the authors on their open study documentation, source code, and SPIRIT checklist.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-425
https://f1000research.com/articles/8-862/v1	13 Jun 19	{ "type": "Opinion Article", "title": "Academia’s Big Five: a normative taxonomy for the epistemic responsibilities of universities", "authors": [ "Rik Peels", "René van Woudenberg", "Jeroen de Ridder", "Lex Bouter", "René van Woudenberg", "Jeroen de Ridder", "Lex Bouter" ], "abstract": "This paper proposes a normative taxonomy by which universities can express the extent to which they meet five core epistemic responsibilities. Epistemic responsibilities are responsibilities that have to do with the attainment of knowledge and understanding. The core epistemic responsibilities, which we call the Big Five, are to (1) foster research integrity, (2) teach for intellectual virtue, (3) address the big questions of life, (4) give humanistic inquiry and education a proper place, and (5) serve society. The paper characterizes the Big Five in some detail and explains why they are core epistemic responsibilities of universities. The paper concludes by describing the steps that should be taken in order to test, amend, and implement the taxonomy.", "keywords": [ "Big question", "epistemic responsibilities", "humanities", "research integrity", "societal relevance", "teaching", "university", "virtue" ], "content": "1. Introduction\n\nIn this paper, we propose a normative taxonomy of what we call the ‘Big Five’ in academia: five core epistemic responsibilities of universities. Epistemic responsibilities are responsibilities that have to do with the attainment of knowledge, understanding, insight, rationality, and explanation. Thus, they are to be distinguished from moral responsibilities – that concern the well-being of humans and animals – and practical responsibilities, such as the responsibility to develop societally useful technologies and effective medical interventions1. For each epistemic responsibility, we distinguish five levels describing the extent to which a university meets that responsibility or strives to do so. Our taxonomy is meant as a tool to assess the degree to which a university meets its core epistemic responsibilities. The format we use is inspired by the Transparency and Openness Promotion (TOP) guidelines2 that journals can use to describe the extent to which they meet the goals of Open Science.\n\nThe taxonomy proposed here is a product of two research projects funded by the Templeton World Charity Foundation: Science beyond Scientism (2013–2016) and The Epistemic Responsibilities of the University (2016–2019)3. The first project explored which questions science can and which ones it cannot address, as well as whether the natural sciences are the only reliable source of knowledge. The second project explored what the core epistemic responsibilities of universities are, given various contemporary challenges, such as hypercompetition, publication pressure, the marginalization of the humanities, and the commercialization of the university. In both projects, philosophers worked in close cooperation with biomedical and social scientists. We present this as work in progress, as a starting point for gaining experience with using the taxonomy and consensus building for a more mature version.\n\nThis paper is structured as follows. First, we provide the taxonomy by specifying academia’s Big Five epistemic responsibilities and detailing five levels of meeting them (Table 1). After that, we argue that these are indeed five core epistemic responsibilities of universities (section 2). Finally, we lay out which future steps we aim to take to test, amend, and implement the taxonomy (section 3).\n\n\n2. A normative taxonomy\n\nOur proposal distinguishes five epistemic responsibilities. We consider each to be of equal importance, so the order in which we present them is not hierarchical. The attainment levels I through V for each responsibility, however, are meant hierarchically: each level presents a more advanced stage of meeting the responsibility at issue.\n\nWe think of these responsibilities as attaching primarily to entire universities. So, in order to meet them, each responsibility should have systematic consequences, throughout the university and its faculties, departments, institutes, or other organizational parts. If a university strives to teach for intellectual virtue at the highest level, for example, students throughout the university should be instructed in what these virtues are and stimulated to cultivate them through virtue-building teaching activities.\n\nThis doesn’t mean that there cannot be an internal division of labour when it comes to meeting epistemic responsibilities: giving humanistic inquiry a proper place will primarily fall on humanities departments, although humanities scholars will teach in other departments and collaborate with scientists in other departments as well when a university strives for level V of this responsibility. Similarly, not all departments and research teams will have to serve society in the same way or to an equal degree. There will be significant differences between, say, the theoretical physicists and the nutrition scientists.\n\nWe will now clarify each responsibility briefly and motivate why it belongs on the list of epistemic responsibilities of universities.\n\n1. To foster research integrity. Research integrity is fostered by getting rid of perverse incentives, stimulating good mentoring, having an open research climate, and so on. Detrimental research practices include both rare major research misbehaviours like fabrication of data and highly prevalent minor misbehaviours like selective reporting4. By ‘responsible conduct of research’ we mean behavior that meets the principles and standards for good research, as laid out in major codes of conduct for research integrity5. Such behavior can be stimulated at the level of individual scholars, but also that of groups, such as research teams or departments. Ideally, research integrity is promoted for both individuals and groups.\n\nThe results of scientific and scholarly research play a crucial role in modern society. Universities carry out a substantial part of this research and educate and train researchers who perform the studies and apply the results. To ensure the validity and trustworthiness of findings research needs to be performed according to the principles and standards for research integrity. In recent years it has become painfully evident that there is substantial room for improvement in the level of compliance to these principles and standards6. Surveys indicate that 2% of researchers admit to having fabricated or falsified data themselves, while one third says that they have been engaged in less serious questionable research practices7. Only 10 – 40% of study results turn out to be reproducible when the study is repeated8. This is due to small sample sizes, selective reporting and other questionable research practices. Although these phenomena are understudied, it is clear that more transparency9 and specifically preregistration10 of study protocols and data analysis plans will lead to considerable improvements. This epistemic responsibility entails not only the adoption of transparency and educating staff and students in responsible conduct of research, but also removing perverse incentives from the ways in which researchers are assessed11 and performing research on research to strengthen the evidence base for optimizing research integrity.\n\n2. To teach for intellectual virtue. Among the intellectual virtues are: open-mindedness, attentiveness, charitableness, intellectual courage, creativity, curiosity, discernment, honesty, intellectual humility, objectivity, parsimony, perseverance, studiousness and wisdom12. Educating for intellectual virtues is part of the traditional ideal of Bildung. Moral virtues, such as generosity, kindness, or benevolence, may well also be important and universities might have a responsibility to cultivate them, but, if so, this isn’t an epistemic responsibility. When universities choose to teach for intellectual virtues, they can do so, minimally, by merely bringing up these virtues in educational settings; or they can explore them through case studies of intellectually virtuous scientists and scholars and their contributions to epistemic progress, or, most advanced, they can actively cultivate intellectual virtues in their students by having them mimic and practise intellectually virtuous behaviour. Developing intellectual virtues requires not merely instruction about virtues or reflection on them, but training and exercise13.\n\nTeaching for intellectual virtues is a responsibility of universities almost by definition. Intellectual virtues are broadly understood as qualities that make someone a well-trained thinker or inquirer14. Hence, the two main tasks of universities, research and teaching, are both served by teaching for intellectual virtue. By aiming to train students to become skilled thinkers (perhaps among other things), universities set an ambitious goal for education. Similarly, academic research also needs skilled thinkers. Moreover, the intellectual virtues are widely relevant outside the university: in politics, journalism, medicine, law, law enforcement, social work – it’s hard to think of any sphere of life where good, analytical, critical, clearheaded, creative, or otherwise high quality thinking wouldn’t matter.\n\n3. To address the big questions of life. By ‘the big questions’ we mean such questions as: What is the origin and ultimate destination of all that exists? What is the future of the earth’s ecosystem? What is consciousness? Do humans have free will? Is there (objective) good and evil? Can the human mind understand the world and, if so, how? Does life have meaning? Does God exist? How does science relate to religion? These are universal questions in the sense that they have been asked in most cultures and societies throughout history, up to the present; they are not restricted to local concerns or specific academic disciplines.\n\nAddressing the big questions of life is an epistemic responsibility of the university for a number of reasons. First, these questions are too important to be left entirely to non-academics. Second, many academics themselves are greatly interested in these questions, even if their specialized science and scholarship does not or even cannot answer them. Third, clarifying and attempting to answer these questions affects how we look at ourselves and what we deem important. Fourth, several big questions are factual questions – highly abstract and large scale, but factual nonetheless. They are not questions about tastes or preferences. They seem to admit of true or false answers. So, at least in principle, they fall within the purview of scientific and humanistic inquiry. Fifth, big questions can inspire smaller and more manageable research questions. For example, asking about the fundamental nature of reality led to the hypothesis of atomism in Ancient Greece15. Ideas about God’s perfection and omnipotence led Galileo to assume that mathematics is our best guide to understanding the orbits of the planets and hence that heliocentrism rather than geocentrism is correct16. Darwin asked whether humans are unique or whether all life on earth is monogenetic, which led him to develop evolutionary theory17. Einstein opposed the Copenhagen interpretation of quantum mechanics on metaphysical grounds, as he didn’t believe the universe could be fundamentally probabilistic18. For these reasons universities cannot and should not operate as if the big questions don’t exist or cannot be taken seriously. Rather, they should take them seriously and mobilize their intelligence as well as state of the art science and scholarship to address these questions in an intellectually responsible way.\n\n4. To give humanistic inquiry and education a proper place. Universities ought to have room for the full range of academic disciplines, in both the sciences and the humanities. Many of today’s most urgent challenges in society cannot be solved by purely scientific or technological means; successful solutions require compelling communication, consideration of moral values and norms, and in-depth understanding of cultures and religions. All of these things, and much more, are studied in the humanities. Hence, universities ought to facilitate and embrace humanistic inquiry and teaching, and stimulate interdisciplinary collaborations between scientists and humanities scholars. Of course, some universities – technological universities, for instance – do not include humanities departments. For them, this responsibility may be either left aside, or be interpreted as follows: the responsibility to give due weight to knowledge and understanding produced by humanities departments in other universities.\n\nGiving the humanities a proper place is a responsibility of the university for one basic reason: the humanities can deliver truth, knowledge and insight in areas where the sciences cannot19. The humanities have their own objects of study: they study objects that have “meaning” in a special sense, viz. meaning that derives from human conventions, from human intentions, and/or from human purposive behaviour20. The knowledge and understanding the humanities provide differs from the knowledge and understanding that the sciences offer, in that the former is often ‘indexical’ (that is, related to human interests and concerns), ‘perspectival’ (it specifies how things look from, say, a romantic perspective), and value-related (that is, related to social, political, moral, aesthetic, or religious values)21. In addition to this, the humanities are particularly suitable for educating students to become well-informed, critical citizens who can reflect on socially urgent questions about life, health, education, justice, equality, liberty, etc. and participate fully in society and politics22.\n\n5. To serve society. Universities can serve society at a number of levels: local (a city or region), national, or international, humanity worldwide. What we have in mind here is serving society epistemically, that is, to help society acquire true belief, knowledge, and understanding about important issues. So, what we have in mind are such things as press releases, expert advice, popular articles, opinion pieces, public lectures, interviews, and so on. Of course, universities sometimes also serve society in a more practical manner, e.g., by way of proposing effective policies, producing medical interventions and other technologies. Such practical interventions are often based on scientific evidence, so the epistemic and the practical are not entirely separate, but they can be distinguished for analytical purposes. We focus on the former here, as our taxonomy concerns the epistemic rather than the moral, practical, or social responsibilities of universities.\n\nThis is a responsibility for at least two reasons. First, many scientific and scholarly discoveries are so complex that if academics do not disseminate their knowledge, those discoveries will remain unknown among the larger audience. Second, it often requires extensive academic knowledge to understand the importance and ramifications of various discoveries. Knowledge and understanding are of intrinsic epistemic value. If the university does not serve society by sharing academic knowledge and understanding, then, for much academic knowledge and understanding, that value will be attained only by a very small group of academics in the relevant field. If the university takes its epistemic responsibility of knowledge dissemination seriously, then much larger groups – academics in other fields, society as a whole – will attain those epistemic values.\n\n\n3. Future steps\n\nAs indicated, we propose our normative taxonomy as a tool to assess the degree to which a university meets the Big Five epistemic responsibilities. Our proposal is a first attempt; in future work we aim to validate, test, amend, and implement the taxonomy. We envision doing this in four consecutive steps.\n\nFirst, we want to fine-tune our taxonomy in a Delphi Study23,24 with international experts that aims in its first round at adding, replacing, and reformulating various epistemic responsibilities. The second and third Delphi rounds will seek consensus on the corresponding levels of meeting the responsibility at issue and explore what the best practices are for reaching higher levels of specific responsibilities.\n\nNext, we will organize co-creation workshops25 with representatives of relevant stakeholders in order to discuss a penultimate version of the taxonomy. The focus of the workshop will be on the operationalization of the levels of meeting the different epistemic responsibilities in a way which makes application of the taxonomy feasible, transparent, and as objective as possible.\n\nThen, we will test and qualitatively evaluate the taxonomy in a number of universities, resulting in a definitive description of the responsibilities, the levels, and a toolkit of best practices.\n\nFinally, we will publish and disseminate the results on a dedicated website and explore whether the taxonomy is a suitable alternative for, or addition to, the currently dominant Academic Ranking of World Universities26 and the Times Higher Education World University Rankings27.\n\n\nData availability\n\nNo data is associated with this article.", "appendix": "Grant information\n\nWork on this paper was funded by the Templeton World Charity Foundation and was carried out within the project The Epistemic Responsibilities of the University (2016–2019). The opinions expressed in this publication are those of the authors and do not necessarily reflect the views of the Templeton World Charity Foundation.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nFootnotes\n\n1Some of us have made this distinction before; see, for instance, Peels R, de Ridder J, Haven T, Bouter L (2019). Value Pluralism in Research Integrity. under review.\n\n2Nosek B et al. (2015). Promoting an Open Research Culture. Science 348 1422–5, https://doi.org/10.1126/science.aab2374 and https://cos.io/our-services/top-guidelines/\n\n3For some of the outcomes, see de Ridder J, Peels R, van Woudenberg R (2018). Scientism: Prospects and Problems (New York: Oxford University Press).\n\n4See National Academies of Sciences, Engineering, and Medicine (2017). Fostering Integrity in Research (Washington, DC: The National Academies Press). https://doi.org/10.17226/21896\n\n5See, e.g., the Singapore Statement (https://wcrif.org/guidance/singapore-statement); ALLEA (2017). The European Code of Conduct for Research Integrity Revised Edition, https://ec.europa.eu/research/participants/data/ref/h2020/other/hi/h2020-ethics_code-of-conduct_en.pdf, last visited May 14th 2019; DORA (2018). San Francisco Declaration on Research Assessment, https://sfdora.org; VSNU (2018). Netherlands Code of Conduct for Research Integrity, Revised Edition, http://www.vsnu.nl/files/documents/Netherlands%20Code%20of%20Conduct%20for%20Research%20Integrity%202018.pdf, last visited May 14th 2019.\n\n6See National Academies of Sciences, Engineering, and Medicine (2017). Fostering Integrity in Research (Washington, DC: The National Academies Press). https://doi.org/10.17226/21896; Hiney, M (2015). Research Integrity: What it Means, Why it Is important and How we Might Protect it. EC Briefing Paper - Research Integrity (https://www.scienceeurope.org/wp-content/uploads/2015/12/Briefing_Paper_Research_Integrity_web.pdf)\n\n7Fanelli, D (2009). How Many Scientists Fabricate and Falsify Research?. PLoS ONE 4(5); e5738. https://doi.org/10.1371/journal.pone.0005738 Note that these numbers concern scientists’ self-reporting; they go up considerably when researchers are asked whether they’re aware of colleagues having committed scientific fraud or engaged in questionable research practices.\n\n8KNAW Replication studies (https://www.knaw.nl/nl/actueel/publicaties/replication-studies); Baker, M (2016). Is There a Replicability Crisis? Nature 533(7604): 452–454. https://doi.org/10.1038/533452a Some of us have recently argued that replication is also a desideratum in the humanities. See Peels R and Bouter L (2018). The Possibility and Desirability for Replication in the Humanities. Palgrave Communications 4:95, https://doi.org/10.1057/s41599-018-0149-x.\n\n9Wilkinson MD, Dumontier M, et al. (2016). The FAIR Guiding Principles for Scientific Data Management and Stewardship. Scientific Data 3: 160018. https://doi.org/10.1038/sdata.2016.18; https://www.go-fair.org/fair-principles/\n\n10Nosek B, Ebersole CR, DeHaven AC, Mellor DT (2018). The Preregistration Revolution. PNAS 115: 2600–2606. https://doi.org/10.1073/pnas.1708274114\n\n11Moher D, Naudet F, Cristea IA, Miedema F, Ioannidis JPA, and Goodman SN (2018). Assessing Scientists for Hiring, Promotion, and Tenure. PLoS Biol 16(3): e2004089. https://doi.org/10.1371/journal.pbio.2004089\n\n12See Battaly H, ed. (2010). Virtue and Vice, Moral and Epistemic (Oxford: Wiley-Blackwell); Baehr J, ed. (2016). Intellectual Virtues and Education: Essays in Applied Virtue Epistemology (New York: Routledge).\n\n13See Ritchhart R (2002). Intellectual Character: What It Is, Why It Matters, and How to Get It (San Francisco: Jossey-Bass).\n\n14See Roberts R and Woods J (2007). Intellectual Virtues (New York: Oxford University Press); Baehr J (2011). The Inquiring Mind (New York: Oxford University Press); Battaly H, ed. (2018). Routledge Handbook of Virtue Epistemology (London: Routledge).\n\n15Berryman S (2016). Ancient Atomism, in: Edward N. Zalta (ed.), The Stanford Encyclopedia of Philosophy https://plato.stanford.edu/archives/win2016/entries/atomism-ancient/.\n\n16See Shea W and Artigas M (2003). Galileo in Rome: The Rise and Fall of a Troublesome Genius (Oxford: Oxford University Press); Remmert V (2005). Galileo, God, and Mathematics. In Koetsier T, Bergmans L (eds.), Mathematics and the Divine. A Historical Study (Amsterdam: Elsevier), pp. 347–360.\n\n17See Desmond A and Moore J (1992). Darwin (New York: W.W. Norton).\n\n18See Isaacson W (2017). Einstein. His Life and Universe (New York: Simon & Schuster).\n\n19Thus also, Peels R (2018). Epistemic Values in the Humanities and the Sciences, History of Humanities 3.1, 89–111. https://doi.org/10.1086/696304\n\n20See van Woudenberg R (2018). The Nature of the Humanities, Philosophy 93: 109–140. https://doi.org/10.1017/S003181911700047X\n\n21See chapter 2 of Foley R (2018). The Geography of Insight. The Sciences, the Humanities, How They Differ, Why They Matter (Oxford: Oxford University Press).\n\n22See Nussbaum M (2010). Not for Profit: Why Democracy Needs the Humanities (Princeton: Princeton University Press).\n\n23Terwee CB, Prinsen CAC, Chiarotto A, Westerman MJ, Patrick DL, Alonso J, Bouter LM, de Vet HCW, Mokkink LB (2018). COSMIN Methodology for Evaluating the Content Validity of Patient-Reported Outcome Measures: A Delphi Study. Quality of Life Research 27: 1159–70. https://doi.org/10.1007/s11136-018-1829-0\n\n24 https://web.njit.edu/~turoff/pubs/delphibook/delphibook.pdf.\n\n25Ramaswamya V and Ozcan K (2018). What is Co-Creation? An Interactional Creation Framework and its Implications for Value Creation. Journal of Business Research 84: 196–05. https://doi.org/10.1016/j.jbusres.2017.11.027\n\n26http://www.shanghairanking.com/\n\n27https://www.timeshighereducation.com/world-university-rankings" }	[ { "id": "49894", "date": "01 Jul 2019", "name": "Britt Holbrook", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article proposes what Peels et al. call a “normative taxonomy” of the “Big Five” epistemic responsibilities universities ought to meet: (1) fostering research integrity, (2) teaching (for) intellectual virtues, (3) addressing the big questions, (4) valuing the humanities, and (5) serving society. This normative taxonomy essentially serves as an analytical rubric designed to assess the performance of universities. One could easily imagine – and in fact, Peels et al. propose as much in §3 – refining the rubric presented in Table 1, using it to assess the performance of various universities, and then comparing their “Big Five” scores to their world university rankings. We agree that undertaking these tasks presents many enticing possibilities, especially to those of us inclined against bean counting approaches to accountability. Nevertheless, since they position themselves as aspiring university rankers, they should address the range of critiques targeting existing rankings, their methodologies, and the performativity of these rankings. They should then situate their own approach within that context, arguing especially that it is an improvement on – and escapes the criticisms levelled at – existing rankings. This is our global concern.\n\nBelow, we offer a number of other substantial concerns, followed by a shorter list of minor issues to consider:\n\nWhy these five?\nPeels et al. propose five epistemic responsibilities of universities as the Big Five. The authors point out that they consider each of the Big Five equally important. They also defend each of the five as indeed responsibilities that universities ought to meet. However, they fail to argue that these five epistemic responsibilities are the most important epistemic responsibilities for universities, that these five are in fact equally important, that these five are sufficient to capture the epistemic responsibilities universities ought to meet, or that universities have no other equally worthy responsibilities that ought to be included.\n\nWhy five?\nWe agree that the proposed Big Five are epistemic responsibilities that universities ought to meet. Using the proposed rubric to evaluate universities might provide valuable information about particular areas of strength and weakness at a given university. However, designing the rubric around five separate epistemic responsibilities suggests that universities could meet each of their epistemic responsibilities to varying degrees. One university might be particularly strong in terms of fostering research integrity but fail miserably at teaching for intellectual virtue. Another might excel at addressing the big questions but do a lousy job of valuing the humanities. Although these possibilities sound prima facie plausible, using an analytic approach to epistemic responsibilities also generates some less than desirable outcomes. Could a university that fails to teach for intellectual virtue really excel at fostering research integrity? That seems plausible only if we have a very limited procedural view of what counts as fostering research integrity. Without valuing the humanities, how could a university excel at addressing the big questions? Unless we adopt scientism, the answer seems to be that it could not. Similar problems arise when we consider the relations between the other proposed epistemic responsibilities. The use of a taxonomical vocabulary further strengthens this worry. Taxonomies should display not only differences, but also relationships and similarities. The current form of representation is not, in that sense, a taxonomy, but (for the most part) a normative list. Only when the ties binding the elements together are conceptualised convincingly can we convincingly speak of a taxonomy. For this reason, we suggest that the authors consider enriching their list to raise it to the level of a taxonomy or abandon talk of a ‘taxonomy’ altogether. The authors could adopt the vocabulary of an analytical rubric, instead.\n\nEven if the authors decide to use the language of rubrics rather than taxonomies, we suggest that the authors consider a more holistic approach along with an analytic one, just as virtue ethics appeals not only to the individual virtues, but also to the question of character. Rather than describing teaching for intellectual virtue as part of Bildung, perhaps the authors could consider fostering the ability to pursue Wissenschaft while cultivating Bildung as analogous to a university’s character. Call it the W-B rubric, under which different (now, no longer separate) epistemic responsibilities could be grouped together to indicate different levels of overall achievement. In order for a university to score best on the W-B rubric, it would have to excel at meeting all of its epistemic responsibilities (for a brief discussion of the difference between analytic and holistic rubrics, as well as examples and further references, see Rubrics: useful assessment tools. Centre for Teaching Excellence, University of Waterloo1).\n\nWhy insist on separating epistemic from other responsibilities universities have?\nThis question follows on the previous one, in that it aims to nudge the authors away from using only an analytic approach. By insisting on distinguishing epistemic responsibilities from moral, practical, and social responsibilities, the authors impoverish the idea of what it means for universities to serve society. If the goal is to work towards alternative forms of assessment and accountability (thereby politicizing them further), excluding other responsibilities and artificially fragmenting the landscape accordingly, one would expect [a] the epistemic responsibility taxonomy to be situated within a larger responsibility taxonomy and/or [b] an argument that leaving other responsibilities aside is preferable. More in line with the suggestion to pursue a more holistic approach, we must ask: could we really say that a university is a good university if it meets only its epistemic responsibilities? We think not. Perhaps the W-B rubric we suggest, or another holistic approach, could help address this issue. Note that one could also include analytical rubrics for moral, practical, and social responsibilities of universities and incorporate the responsibilities included in each of these separate rubrics into a holistic approach.\n\nOur point is not that analysis is always bad. Indeed, analysis can be quite informative and useful as part of a formative evaluation. We are simply suggesting that the authors consider using their analytic approach to complement a more holistic approach.\n\nWho counts, and why do they count?\nIn §3, the authors suggest optimising their rubric by means of a Delphi Study “with international experts”, which could lead to fine-tuning it, but possibly also to radical revisions. If the latter is the case, then what is the status of the current five epistemic responsibilities? When it comes to the actual Delphi study, whom they choose to participate in the study will likely make a very big difference to the final design of the rubric. Presumably, the authors focus on “experts” because they assume experts know more about epistemic responsibilities than non-experts do. Yet, the way in which the authors present it suggests a retreat to the deficit/diffusion model of public understanding of science. Not only has the deficit model been shown to be factually incorrect, it also presupposes a social contract for science and scholarship that imagines universities as ivory towers. In their discussion of a university’s responsibility to serve society epistemically, the authors focus on the supply side of knowledge production, suggesting that knowledge dissemination is how best to serve society. Arguably, however, serving society – even if we limit this to an epistemic responsibility – means something other than telling society what we academics think they need to know. Here again, the separation between epistemic and other responsibilities creates a lot of friction, since a departure from the deficit model requires a high degree of interaction and participation beyond universities. In fact, one could argue that interaction and participation are epistemic requirements (co production of knowledge).\n\nAlong these lines, we suggest that non-experts may have valuable feedback to offer, even if the authors ultimately decide only to pursue the development of an analytic rubric for epistemic responsibilities of universities. One way to include the demand side of knowledge production in the design of their rubric would be to recruit non-expert stakeholders to participate in the proposed Delphi Study and workshops or organise parallel expert (Delphi) and citizen (Citizen Summit) consultations.\n\nWe also have a few more minor points that nevertheless warrant attention:\n\nDespite the argument that the rubric is to be applied to entire universities, the responsibilities focus very much on individuals and groups (which seem to be multiple individuals in the ways in which they are discussed) and less on the level of structures and collectives.\n\nPeels et al. frame irreproducibility solely as the result of sub-par science and thus as a research integrity issue. Their previous work, as well current scholarly debates on the characters and qualities or irreproducibiluty and irreplicability, takes up a much more nuanced position. Perhaps the authors will consider adding some of that nuance here.\n\nExcusing a few universities (technical universities or polytechnics, in this case) of taking responsibility for one of the epistemic responsibilities (#4) suggests that giving humanistic inquiry and education a proper place is optional. This seems to conflict with the idea that all responsibilities are equally important. It also opens up possibilities for policies that deprioritise humanities research and teaching (cf. the current Van Rijn report in NL).\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "5678", "date": "06 Jul 2020", "name": "Rik Peels", "role": "Author Response", "response": "We thank the reviewers for their helpful comments and suggestions. Below, we reply to them. Relation to Other Rankings Holbrook and Penders (H&P) urge that we do two things. First, that we situate our taxonomy in the wider context of other rankings. Second, that we address criticisms of existing rankings, and explain why we think ours is an improvement. H&P are right that there are many other rankings, even many other non-standard rankings that are not merely cashed out in terms of publications. Yet, many of them are based on quantitative data, such as (i) number of students; (ii) granted research proposals; (iii) Nobel prize winners; (iv) citation indices; (v) the result of surveys on the opinions on stakeholders with respect to e.g. the reputation of universities. Our proposal focuses on epistemic responsibilities. Whereas other rankings are mainly based on quantitative data and opinions of stakeholders, the instrument we propose is based on mainly qualitative data, and will lead to an ordering in normative categories. Because of these differences, it isn’t immediately clear that extant criticisms of other rankings would apply to our proposed taxonomy. But we’ll look into this more systemically in future work as part of a new project on Epistemic Progress in the University (also funded by the Templeton World Charity Foundation). We explain this in the introduction and the section on Future steps. Why These Five? H&P note that we don’t argue (a) that our five are the most important areas of responsibility; (b) that they are equally important; (c) that they are sufficient to capture the epistemic responsibilities of universities; (d) that universities have no other equally worthy responsibilities. As to (a)–(c), H&P are right that more support is needed to back up these claims – the current paper lays out a proposal based on a survey of literature about epistemic responsibilities of the university and our own analysis thereof. But providing further support is exactly what we aim to do in follow-up work that’s part of the abovementioned new project. As to (d), universities indeed have other, equally important responsibilities. Among them are moral responsibilities (like providing safe environments for students and faculty, and taking care of the wellbeing of human and animal test subjects, treating indigenous people fairly); legal responsibilities; financial responsibilities; social responsibilities (like producing useful technologies and effective medical interventions), and more. What sets universities apart from many other organizations and institutions is that their main goals are epistemic in nature: their primary aims are to produce and disseminate knowledge and understanding. That is why we focus on these. We now make this more explicit in the introduction. The Relations between Various Values H&P say that the values are interrelated (if there is research integrity, there will or must be such a thing as teaching virtue; and how can universities address the Big Questions without cultivating the humanities?) and that we do not acknowledge this fact. And this, they maintain, undermines our goal of offering a taxonomy—for a taxonomy “should display not only differences but also relationships and similarities”. Instead of offering a taxonomy, they urge, we could also offer rubrics. We agree that the epistemic responsibilities we distinguish are related to each other. For instance, responsibility 1 (to foster research integrity) surely has to do with responsibility 2 (to teach for intellectual virtue): it would be highly surprising if a university is serious about research integrity but does not teach for curiosity, open-mindedness, thoroughness, and intellectual perseverance. However, this doesn’t mean that we cannot draw analytic distinctions between the responsibilities that allow us to focus on different parts and aspects of universities’ epistemic responsibilities. It may not always be possible (or even desirable) in practice to implement these responsibilities through separate activities, interventions, or policies, but they can be distinguished through an analytical lens. We consider that an advantage of our approach. Furthermore, the exact relations between these responsibilities is up for debate, so we wanted to leave room for different opinions about the relations between these responsibilities while working towards more consensus that these are indeed key epistemic responsibilities of universities. We have added this reasoning at the end of section 2. As to the suggestion of explicating relationships and dependencies and developing a rubric rather than a taxonomy, we are happy to take these ideas into consideration as we continue the development of this proposal in future work. A More Holistic Rubric? H&P add that just as the virtues of an individual person are expressions of that person’s character, so the university has (or should have) a character, and their friendly suggestion is that the character of the university is To pursue Wissenschaft while cultivating Bildung. We are sympathetic to this suggestion; universities can have relatively stable dispositions to pursue particular values reliably. We might call this the university’s character or, alternatively, its organizational culture. We take it, though, that this suggestion is already covered by our proposal. It is an epistemic responsibility of universities to give the humanities a proper place next to the natural and social sciences. Our focus on intellectual virtues, moreover, is very much in line with the ideal of Bildung. Deficit/diffusion Model of Public Understanding H&P say that we rely on the incorrect deficit/diffusion model of science communication: the public has a deficit of knowledge, and science communication aims to diffuse the deficit by providing knowledge. We certainly didn’t mean to assume this model, although we can see how our focus on knowledge dissemination under the epistemic responsibility of serving society might give the impression that the role of universities merely is to better inform the public. To prevent this misunderstanding, we have added a new paragraph where we explicitly state that the responsibility of serving society can be a two-way street, where citizens can offer valuable input on which research questions to pursue, how to best pursue them, and how to communicate and use research findings. In some cases, the production of scientific knowledge can even amount to co-creation between scientists, stakeholders, and citizens. The suggestion to include citizens and various stakeholders in proposed Delphi studies is a valuable one, that we will take onboard. Smaller Points H&P raise three smaller points: We focus, they say, more on individuals than on structures and collectives. We’re not sure what gave rise to this impression – as we think of them, all five responsibilities really are responsibilities of the university as a whole, so both its leadership and the individuals affiliated with it, as well as its organizational structure, policies, incentives, etc. We are keenly aware that meeting these responsibilities is as much an issue of individual action as it is of collective action, formal and informal organizational structures and the incentives they give rise to. And, in fact, even broader structures and systems in society. In developing our proposal further, however, we will make sure to keep both the individual and the organizational level in focus. In this paper we suggest that irreproducibility is a sign of sub-par science, so H&P say, and they suggest that we include in a further version of our paper our more nuanced (and already published) views. We’ve done this in the revised version of the paper (note 9). We make it seem as if the humanities are optional. This may be a misunderstanding. All we wanted to do is acknowledge the existence of technical universities, which may not need fully-fledged humanities departments. We do emphasize that they too have responsibilities in addressing the big questions." } ] }, { "id": "57012", "date": "11 Dec 2019", "name": "Jennifer Byrne", "expertise": [ "Reviewer Expertise Molecular biology", "molecular genetics", "human tissue biobanking", "research integrity" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting manuscript which is somewhat outside my field of expertise, which is centred around molecular biology and genetics. There is no doubt that this will have limited my ability to provide a detailed critique of the arguments presented, and this may also limit the value of some of my suggestions. Nonetheless I advance these in the hope that my comments will indicate how this article was received by someone outside the field, given that this paper will interest a broad range of academics and researchers.\n\nMy main comments pertain to the taxonomy proposed in Table 1. I would not argue with the five epistemic responsibilities put forward; however, I did wonder whether five levels of taxonomy are required for any or all of the five proposed responsibilities. Some of the descriptions provided for levels I and II seem unlikely to occur in practice, and I could not always see clear distinctions between some levels proposed, most frequently for levels I and II.\n\nFor example, for “1. To foster research integrity”, I could not see a clear difference between “Issues of detrimental research practices and responsible conduct of research are neglected” (level I) and “Issues…. are occasionally addressed” (level II). There may not be much difference in practice between something being “neglected” or “occasionally addressed”, and it is also not clear how “occasionally” would be reliably measured. Similarly, I could not see a clear difference between level III “Detrimental research practices are addressed… but there is no academic climate that actively stimulates responsible conduct of research” and level IV “There is an academic climate that detects and acts upon detrimental research practices and actively stimulates responsible conduct of research, but only at the level of individual researchers”.\n\nFor “2. To teach for intellectual virtue”, I couldn’t see clear differences between level I “the university…. pays no attention to intellectual virtues” and level II “intellectual virtues are considered important, but not taught”. I also could not imagine any University that would fall into either of these categories.\n\nAgain, I couldn’t see clear differences for the proposed levels I and II for “3. To address the big questions of life”, ie these questions being “neglected” (level I), versus “mentioned but discarded” (level II).\n\nFor “4. To give humanistic inquiry and education a proper place”, I also saw no real distinction between the humanities being “marginalized” (level I) and “not marginalized but considered and treated as being inferior” (level II). Levels III, IV, and V commonly refer to humanities being given a “proper place” in the university, but this definition will naturally vary between universities according to their type, i.e. technical universities versus universities with a strong and stated focus upon the humanities. Some wording within levels III-V also lacked definition, i.e. “in isolation” (level III), “some other disciplines” (level IV), and “across disciplines” (level V). “Disciplines” can be defined quite narrowly by some universities, so working with other disciplines or across disciplines does not always imply working between the humanities and the sciences.\n\nFinally, for “5. To serve society”, universities where “research and teaching are confined to …purely academic challenges” (level I) could argue that they are serving society in this way, possibly by pursuing academic challenges that can indirectly linked to the big questions of life. It also seems unlikely that any university would be defined by level II “Research and teaching identify societal challenges, but the university leaves it to others to confer knowledge … relevant to those challenges”.\n\nAre all factual statements correct and adequately supported by citations?\n\nContemporary challenges such as hypercompetition, publication pressures, marginalisation of the humanities and commercialization of universities (page 3, paragraph 2) should be supported by references.\n\nThe status of reference 1 (footnote 1) which is a manuscript by Peels et al, could be updated (page 3).\n\nAre arguments sufficiently supported by evidence from the published literature?\nAlthough the manuscript states that “more transparency and specifically pre-registration of study protocols and data analysis plans will lead to considerable improvements (in the use of small sample sizes, selective reporting and other questionable research practices, from the preceding sentence), this seems to be somewhat of an overstatement. Firstly, more transparency and pre-registration may not impact on the use of small sample sizes, as these might represent feasibility constraints that pre-registration may not overcome. Secondly, I understand that there is not yet a substantial body of literature that describes the possible benefits and/or drawbacks of study pre-registration, by comparing the results and outcomes of pre-registered studies versus comparable studies that lacked pre-registration.\n\nFinally, the manuscript states that (the big questions of life) “are too important to be left entirely to non-academics”. This statement could be written in a more inclusive way, for example by indicating that as these questions are so important, they are everyone’s concern, including the concern of academics.\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? Yes", "responses": [ { "c_id": "5679", "date": "06 Jul 2020", "name": "Rik Peels", "role": "Author Response", "response": "Jennifer Byrne (JB) first notes that the distinctions between the different levels of meeting our responsibilities remain somewhat unclear. This is a good and important point; these levels ought to be distinguished more clearly. In fact, this is one of the main goals of our new project entitled Epistemic Progress in the University (2020-2023) that was recently funded by the Templeton World Charity Foundation. JB’s remarks provide plenty of food for thought on the distinctiveness of the levels as we formulated them. We are very grateful for that and will certainly take these suggestions into account while developing the taxonomy further. We now mention the new project in the introduction and state that we need to do more work on the distinctiveness of the five levels postulated for each epistemic responsibility. Second, we have updated the publication status of footnote 1 and added references to substantiate what we say on hypercompetition, publication pressure, marginalization of the humanities, and commercialization of universities. Third, JB thinks that we overstate the benefits of pre-registration. We partly disagree. We wrote: “Although these phenomena are understudied, it is clear that more transparency and specifically preregistration of study protocols and data analysis plans will lead to considerable improvements.” We didn’t consider this to be a particularly strong claim, but we have now changed it into: “Although these phenomena are understudied, it is to be expected that more transparency and specifically preregistration of study protocols and data analysis plans will lead to improvements.” Finally, we have adopted JB’s suggestion for a more inclusive formulation about whose concern the big questions are. The text now reads: “The big questions are everyone’s concern, academics included.”" } ] } ]	1	https://f1000research.com/articles/8-862
https://f1000research.com/articles/9-677/v1	06 Jul 20	{ "type": "Research Article", "title": "A Discrete-Choice Experiment to capture public preferences on the benefits of home-visiting programme for teenage mothers", "authors": [ "Eugena Stamuli", "Gerry Richardson", "Michael Robling", "Michelle Edwards", "David J. Torgerson", "Kerry Hood", "Julia Sanders", "Sarah Ronaldson", "Gerry Richardson", "Michael Robling", "Michelle Edwards", "David J. Torgerson", "Kerry Hood", "Julia Sanders", "Sarah Ronaldson" ], "abstract": "Background: Complex health and social care interventions impact on a multitude of outcomes. One such intervention is the Family Nurse Partnership (FNP) programme, which was introduced to support young, first-time mothers. Our study quantified the relative values that the general public place on the outcomes of FNP, as they were identified and measured in the relevant randomized trial, Building Blocks trial (BBs). Methods: A discrete choice experiment (DCE) was employed. Respondents chose between two scenarios describing hypothetical sets of trial outcomes. BBs compared FNP care for teenagers expecting their first child with standard NHS care. 14 attributes covered three areas: pregnancy and birth, child development and maternal life course. Due to large number of attributes, a “blocked attributes” approach was adopted: the attributes were split across four designs which contained two common attributes. Data were analysed separately for each design as well as pooled across four designs. Random effects probit model was employed for the analysis. Results: Over 1000 participants completed four designs. The analyses on the separate designs and those on pooled data yielded broadly similar results. Respondents valued higher the outcomes related to child development and their needs, followed by the outcomes related to maternal life course. Preferences varied by the age of the respondents but not by their guardianship/parentship status. Conclusions: Individual preferences were consistent with a priori expectations and were intuitive. The DCE results can be used to incorporate the general public preferences into the decision making process for which public health and social care policies should be adopted.", "keywords": [ "Family Nurse Partnership", "Building Blocks trial", "maternal and child outcomes", "discrete choice experiment", "random effects probit" ], "content": "Abbreviations\n\nA&E: Accident and emergency; BBs: Building Blocks; DCE: Discrete Choice Experiment; FNP: Family Nurse Partnership; QALYs: Quality Adjusted Life Years.\n\n\nIntroduction\n\nBecoming a mother at a young age is associated with disadvantages for both the mother and her baby1–3. The spectrum of possible disadvantage includes poorer birth and developmental outcomes for children4–6 to reduced life chances in terms of education and employment opportunities for younger mothers7,8.\n\nThe Family Nurse Partnership (FNP) is an intervention designed to improve outcomes for young mothers and their children by supporting families on issues related to health during pregnancy and parenting skills, and on life skills including how to become self-sufficient. The programme has been supported by the Department of Health in many sites in England since 20069 and was evaluated via a randomized controlled trial10,11. The Building Blocks (BBs) trial evaluated four primary outcomes which were birth weight, tobacco use in pregnancy, emergency attendances (A&E) and/or admissions of children within two years of their birth, and the occurrence of subsequent pregnancy within two years of first birth. There was also a range of secondary outcomes, both health and non-health related, which are fully described in the trial protocol10 and report11. The medium-term impact of the intervention is being evaluated through routine data linkage study12.\n\nThe level of success of this intervention, as measured by the outcomes of this trial and the economic evaluation, helps inform commissioners’ decisions on the future of the programme. The nature of the outcomes (health and non-health related), as well as the subject of interest (mother or child related outcomes), add new dimensions to the economic evaluation of such a trial. A cost-utility analysis will present, for example, the cost per mother’s Quality Adjusted Life Years (QALYs) but will fail to include the other outcomes13. A cost-consequences analysis approach reflects the fact that a complex intervention is associated with an array of health and non-health benefits that cannot be measured in a common unit14. Given the disparate nature of relevant outcomes, and the fact that outcomes moved in different directions, it was deemed necessary to establish some relative value (or trade-off) between these outcomes. In addition, given that the public involvement in health-care decision making is an objective of the health care policy makers in the UK15,16, it is important to elicit the preferences of the general public over the different outcomes of the trial, i.e. how the general public weighs up these outcomes. The findings of the randomized trial together with the valuation of the outcomes, as measured in this exercise, provides decision makers with useful knowledge that may inform the judgment about whether the intervention should be adopted or extended on a larger scale.\n\nThe aim of this study was to quantify the relative values that the general public place on outcomes/domains examined in the BBs trial using a discrete choice experiment (DCE). The DCE approach is a well-established method to explore preferences in health services research17,18. The approach is based on asking respondents to state their preferred option between two or more hypothetical scenarios which describe a service or a set of outcomes. The hypothetical scenarios include the descriptors (or attributes) the levels of which can be altered in each choice task. With a sufficient number of scenarios and respondents, it is possible to model the impact of specific descriptors on the choice of the respondents. As the individual considers simultaneously a number of attributes, DCEs are thought to overcome some of the problems that other choice methods, such as ranking, rating or standard gamble, pose19,20.\n\n\nMethods\n\nThe DCE was designed as a web based survey, following the general guidelines and steps for conducting a DCE17,18,21.\n\nThe selection of the DCE attributes and levels drew on the outcomes that were measured in the BBs trial. The trial focused on three areas of research: pregnancy and birth, child health and development, maternal life course and self-efficacy. There were four primary outcomes: tobacco use at late pregnancy (34–36 weeks’ gestation), birth weight, emergency attendances and hospital admissions for the infant within 24 months of birth, and the proportion of women with a second pregnancy within 24 months post-partum. In addition, a large number of secondary outcomes (over 60) were measured across the three aforementioned domains. All four primary outcomes and ten secondary outcomes were included as DCE attributes. The secondary outcomes were selected via a voting process by the team that designed the study protocol of the randomised trial, based on what they perceived as most important efficacy measures.\n\nAll the attributes, with the exception of mother’s health, had two levels (Table 1) describing whether one outcome was positive or negative. This binary format was chosen with the aim to keep the task simple and manageable for the respondents. Mother’s health was described as a four-level attribute, corresponding to the following 3-level EQ-5D health states: 11111, 11112, 11211 and 11212. The focus was only on two of the five domains of the EQ-5D questionnaire22: usual activities and anxiety & depression, as these were considered most likely to be affected in this group of an otherwise healthy population. Further, the attribute was transformed into a continuous variable23 by applying the relevant utility scores i.e. 1, 0.848, 0.883 and 0.812 respectively, based on the UK tariff24.\n\nDue to the large number of attributes, the experimental design was based on the “blocked attribute” approach25,26. The method allows for a large number of attributes to be allocated across more than one design with overlap: some attributes are treated as “common attributes” and appear in all the designs, while the non-common attributes will appear in separately in each design. The fundamental assumption is that the respondents will have the same preferences over the whole set of attributes if they are presented together compared with if they are presented in blocks25. The analysis is based on the “pooled” data from all the designs. The chosen attributes were split across four designs (Table 2) with two common attributes based on the primary outcomes of the trial: one primary outcome related to the mother and one related to the child. The rest of the attributes were allocated to the four designs, attempting to achieve a balanced representation of mother and child related outcomes in each design.\n\nThe designs were constructed in SAS software by using the D-efficiency criterion27. The D-efficiency based designs are the result of the trade-off between degrees of orthogonality and balance, where one or more parameters cannot be estimated when D-efficiency is 0 or the design is perfectly balanced and orthogonal when D-efficiency is 100. Fifteen choice-sets were constructed for the designs A-C and sixteen choice-sets for the design D. These were fractional factorial designs, with only the main effects included. The D-efficiency was 98.96% for designs A-C and 83.91% for design D.\n\nOnce the choice-sets were constructed, qualitative interviews with 15 members of general public were conducted at the Centre for Trials Research, University of Cardiff with the aim to establish whether:\n\nThe respondents understood the choice context and the task in general\n\nThe choice of attributes and levels was appropriate\n\nThe complexity of the task affected the completion of the choice task\n\nThe length of the survey was likely to affect the response rates\n\nTwo rounds of cognitive interviews were conducted. During the first round, a questionnaire was devised using a sample of scenarios from each questionnaire and an extra scenario that simplified the attribute about mother’s health. These questionnaires were tested on six participants. During the second round, four complete questionnaires were used (designs A to D28) and were tested on eight participants.\n\nThe feedback from this piece of work resulted in a few changes to the wording of the original attributes, but no changes in the experimental design.\n\nThe study was a web based, self-completion survey to generate good response rate whilst remaining relatively inexpensive. A market research company (Dynata, former Research Now) undertook the respondents’ recruitment, data collection and handling. The survey was translated into an online, web-based questionnaire which could be accessed by the respondents via their computers. Members of the UK general public, across various geographical locations, who were ≥18 years old were invited via email and text messages to respond to the survey and reimbursed GBP 2.5 for their participation. Probabilistic sampling was utilised with the aim to achieve population-representative sample from the online panel of the market research company. Data were collected on a number of sociodemographic characteristics to assess whether these characteristics have an impact on the choice of the preferred outcomes.\n\nFour groups of respondents were randomly allocated to complete one of the four designs (A to D) and a fifth group completed all four designs. A minimum of 70 respondents per design was estimated based on the following formula29:\n\nWhere:\n\nc is the number of analysis cells, or the largest number of levels for any attributes, for a design which includes only main effects, t is the number of choice tasks and a is the number of alternatives.\n\nFollowing an introductory, informative page, respondents were asked to choose the most preferred scenario out of two in each choice set describing possible outcomes of the trial.\n\nDemographic characteristics of the sample were compared with the general UK population (% representation for each variable) to gauge whether the sample was a good representation of the general population. Data from the DCE were analysed using a random effects probit model in STATA (version 12). This is one of the conventional approaches for analysing DCE data17. The models were estimated on the data from each separate design as well as on the pooled data, with and without interaction with demographic variables.\n\nThe model used for the analysis of the data from the each separate DCE design was:\n\nThe data from the four designs were pooled, where all the attributes were included. For pooled data model, \"no difference\" in attribute levels for the omitted attributes was assumed, and so the variables in the analysis were zeroed out.\n\nSeveral separate regression models were estimated. The first model included only the attributes (i.e. no dummy variables for the different designs or interaction terms with the socioeconomic variables). A separate model was fitted to the pooled data by adding dummy variables for each design. Another model was fitted by including a dummy for the respondents who completed all four designs. Additional models examined how preferences varied according to respondents’ demographic characteristics (i.e. age, gender and parenthood/guardianship status). These models included the fourteen attributes, pooled data from four designs, and interaction term variables.\n\n\nEthics approval and consent to participate\n\nEthics approval was obtained from the Research Governance Committee at the Department of Health Sciences, University of York.\n\nParticipants consent was inferred once the participant read the information sheet30 and proceeded with the questionnaire completion.\n\n\nResults\n\n207 respondents completed all four DCE designs (A+B+C+D), 200 respondents completed one of the A, C or D designs, and 201 completed design B. The sample achieved a good representation of the general adult UK population for all four categories of age, home ownership and marital status but not for gender, as there was an over-representation of female participants (Table 3).\n\naRefers to the percentage of each group for the UK general population above 18 years old; NRS: national readership survey.\n\nThe results from each design separately, as well as the results from the pooled data, with and without including design dummy variables, are presented in Table 4. The design-specific dummy variables are not significant, suggesting that there were no unobserved differences between the samples that completed each design or between the designs. The differences in respondents’ characteristics might have been accounted for by the random allocation of the respondents across designs.\n\n* p<0.05, p<0.01, * p<0.001\n\nThe results of the model on the pooled data, without including the design dummies are discussed here. Given the coding of the binary attributes (Table 1), the negative signs of the coefficients would suggest that respondents’ utility decreases as the level of the attribute increases, or becomes a negative outcome (0= positive outcome, 1=negative outcome). Positive signs would indicate the contrary i.e. respondents derive higher utility as the level of the attribute decreases (i.e. becomes a “positive outcome”). All the regression coefficients have the expected signs and are statistically significant, indicating that the respondents prefer a programme that would improve the outcomes described in this exercise. The results are in line with the a priori expectation that positive outcomes related to vulnerable, pregnant women and their children are preferable to negative outcomes.\n\nThe highest ranked coefficients for the separate designs are the five top highly ranked attributes in pooled data model, showing a consistency of results across models. The relative ranking of the two common attributes (second pregnancy and A&E attendances) is the same in all the models: A&E has a larger coefficient than occurrence of second pregnancy, showing that it is always considered a more important outcome than the occurrence of second pregnancy. In the pooled model, the largest coefficients are mainly related to outcomes that affect children directly: whether the mother fulfils the child’s needs, whether there is a bond between mother and child, A&E attendances for the child and the language development. Therefore, one could conclude that the outcomes related to the child are valued higher than the outcomes that are related to the mother.\n\nThe models based on interaction effects with demographic characteristics (Table 5) demonstrate that preference for the occurrence of child’s A&E attendances, education and employment of the mother and whether the child’s needs are met, varied by gender. The preferences between genders change in terms of their magnitude rather than the nature of the outcome. Women have higher disutility than men if the above attributes take the “negative outcome” value, i.e. the child attends A&E, the mother does not return to education or employment, and the child’s needs are not met.\n\n* p<0.05, p<0.01, * p<0.001 *demographic refers to each demographic characteristic included in the analysis i.e. gender, age, guardianship\n\nPreferences varied by age with significant differences observed for A&E attendances, smoking during pregnancy, employment of the mother, vaccination of the child and mother’s confidence. For child’s A&E attendances, vaccination and mother’s confidence, the working-age respondents (age <= 65) derive higher utility from the negative outcomes taking place (i.e. children attends A&E during the first two years of their life, they do not have the recommended vaccinations, and mother does not feel confident that she can achieve her goals) compared to the older respondents (age > 65).\n\nPreferences did not vary greatly by guardianship status. Differences were observed for birth weight and language development of the child. Parents/guardians received higher utility if the birth weight of the baby was below a healthy range and the language development of the child was not according to its age.\n\n\nDiscussion\n\nThis DCE has established the relative values that the general public places on the outcomes measured in the BBs trial. Over 1000 responses were collected from a sample that was broadly representative of the UK general public on key variables. The large sample is one of the strengths of this study as it enhances the statistical efficiency. The guidelines on conducting DCEs suggest that sample sizes in the range of 1000 to 2000 respondents will produce small confidence intervals, even if the experimental design is not particularly efficient33. This is particularly important in our study where the data were pooled from four designs and the statistical efficiency might have been compromised. Respondents were randomly allocated to complete one or all four designs to minimize the selection bias.\n\nThe study shows that the general public value more highly outcomes that affect children directly: whether the mother fulfils the child’s needs and the bond she develops with the child, child’s A&E visits, and their language development. Mother’s smoking behaviour during pregnancy and the relationship she has with her partner are also ranked high in the preference of the general public. Outcomes directly related to the mother, such as employment, education or her confidence in achieving goals or solving problems, are ranked much lower. In general, the results are intuitive, even more so when gender is taken into account. Women value the outcomes related to the mother higher than men. Although the direction of the preferences for both age and guardianship status appear counterintuitive, there are possible explanations for this. For example, parents may be more acceptant of variability and less rigid about what is considered “normal”.\n\nThe data from the four designs were pooled by the use of two common attributes based on the assumption that the respondents’ preferences for the 14 attributes would be the same regardless of whether they are presented all together or in separate designs25. This assumption would have led to the same coefficients for the common attributes in all designs. Quantitatively, this was not the case as the beta coefficients for the common attributes were different. Still, the ranking of the common attributes was the same across the four designs. This was confirmed in the pooled data: respondents value the attribute “A&E attendances for children” as being more important than the occurrence of second pregnancy. Also, the top five ranked attributes in the pooled model are the top ranked attributes in the separate designs. This provides reassurance that pooling the data would not produce misleading results.\n\nThis DCE included a broad range of outcomes, related to both the mother and the child. The advantage of including a large number of attributes lies on the fact that, by mirroring the outcomes measured in the BBs trial, one broadens the decision making context, where outcomes for different subjects (mother and child, in this case), and both health and non-health outcomes can be incorporated in the trading-off process. The probability of the uptake of the FNP can be estimated by combining the information on the effectiveness of the trial (i.e. which of the outcomes are improved by this intervention) with the preference weights for these outcomes.\n\nHowever, there are a number of caveats. The choice task given to the respondents was somewhat simplified to make the task more approachable to lay persons. The attributes were presented in a binary format and no attempt was made to assign levels other than describing the attribute as a ‘positive’ or ‘negative’ outcome. This approach has led to the preference values being calculated for fairly broad attributes, which could be argued, do not reflect the nuanced outcome differences seen in the trial. An ideal scenario would have been to assign more levels, reflecting the outcomes observed in the trial. However, there were two reasons for adopting this approach: firstly, the nature of the research question might have been too specific for a proportion of the respondents. For example, people without children might have found it more difficult (e.g. the task is more abstract) to value the importance of certain child-related outcomes. Secondly, the large number of attributes called for a simple approach to keep the task manageable and avoid cognitive burn-out of respondents. As an additional limitation, while respondents were able to trade-off attributes, it is not clear whether there are interactions between attributes that have been omitted from the analysis. Omission of important interactions may lead to biased parameter estimates, i.e. an under or overestimation of the relative value of an attribute.\n\nImportantly, in order to be consistent with decision making bodies, such as NICE, the DCE responders were members of the general public. However, there is also an argument for considering the preferences of individuals who have experience in the condition of interest. Brazier et al. (2005)34 call for greater debate on the subject of patients’ valuation of health states. DCEs based on, for example, recent teenage mothers may yield differing estimates of the relative importance of attributes.\n\nDespite these limitations, this DCE can be used to place the results of the BBs trial in a decision-making context, while taking into account the views of the general public for the preferred benefits or outcomes of FNP. An illustrative example for such an approach included two extreme scenarios where the BBs trial results were compared with a best and a worst-case trial output. The BBs trial demonstrated that the FNP results in small improvements in three of the secondary outcomes that were included in the DCE. No improvements were observed in the primary outcomes. This in turn, is translated into an extremely small probability of the FNP being accepted as a public health policy programme by the general public compared to the best-case scenario, where all the outcomes included in this DCE are improved by the intervention. Naturally, the probabilities of acceptance would change depending on what outcomes are being improved by the intervention but also on how highly these have been valued by the general public. Hence, a scenario where, for example, the trial demonstrates improvements in all the primary outcomes (therefore, the intervention is considered a successful one) but these are not valued highly by the general public resulting in a low probability of acceptance, would be entirely plausible. Nevertheless, the DCE provides a decision-making tool when the contribution of the general public or any other specific group of respondents is considered important.\n\n\nConclusion\n\nWhere complex public health interventions are expected to impact upon a range of potential beneficial outcomes, understanding how to use evidence to inform policy becomes even more important. Home visiting programmes, such as the Family Nurse Partnership (FNP), have been evaluated against a wide range of outcomes (clinical and non-clinical) both in the short-term, such as in the Building Blocks trial, and also in the much longer-term (such as in the US trials of NFP35–37). There has also been some academic and policy debate38–40 following publication of the main trial results of FNP in England11 about the relative importance of the primary and secondary outcomes selected when commissioning the trial, which itself was only ever able to report on short-term benefit. For example, the two maternal primary trial outcomes were smoking in pregnancy and a second pregnancy within two years following the first child’s birth. The former but not the latter were rated as of high value to respondents in the DCE and both are subject to further development work by the FNP programme itself post trial publication41. A greater understanding of the importance of a short inter-pregnancy interval in the relevant English context and how policy should respond remains an important task.\n\n\nData availability\n\nHarvard Dataverse: FNP Discrete Choice experiment, https://doi.org/10.7910/DVN/DUL9KT42.\n\nHarvard Dataverse: Designs A-D, https://doi.org/10.7910/DVN/GK4TPV28.\n\nHarvard Dataverse: Patient information sheet https://doi.org/10.7910/DVN/JBVRTZ30.\n\nHarvard Dataverse: Variables key https://doi.org/10.7910/DVN/L8QOKC43.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Acknowledgments\n\nWe thank Julia Witt, Assistant Professor at University of Manitoba, for the email communication which helped to clarify methodological points.\n\nThis study was presented as an abstract at the Fifteenth Annual International Conference on Health Economics, Management & Policy 20–23 June 2016, Athens, Greece (abstract 25, pg 50).\n\n\nReferences\n\nBonell C: Why is teenage pregnancy conceptualized as a social problem? A review of quantitative research from the USA and UK. Cult Health Sex. 2004; 6(3): 255–72. PubMed Abstract \| Publisher Full Text\n\nSocial Exclusion Unit. Teenage Pregnancy-Report to Parliament. London: Her Majesty’s Stationery Office. 1999. Reference Source\n\nDepartment for children schools and families and Department of Health. Teenage pregnancy strategy: Beyond. 2010. Reference Source\n\nParanjothy S, Broughton H, Adappa R, et al.: Teenage pregnancy: who suffers?. Arch Dis Child. 2009; 94(3): 239–45. PubMed Abstract \| Publisher Full Text\n\nChen X, Wen S, Fleming N, et al.: Teenage pregnancy and adverse birth outcomes: a large population based retrospective cohort study. Int J Epidemiol. 2007; 36(2): 368–73. PubMed Abstract \| Publisher Full Text\n\nLópez Turley RN: Are children of young mothers disadvantaged because of their mother’s age or family background? Child Dev. 2003; 74(2): 465–74. PubMed Abstract \| Publisher Full Text\n\nErmisch J: Does a ‘Teen-birth’ have Longer-term Impacts on the Mother? Suggestive Evidence from the British Household Panel Study.Working Papers of the Institute for Social and Economic Research, paper 2003-32, Colchester: University of Essex. 2003. Reference Source\n\nHarden A, Brunton G, Fletcher A, et al.: Teenage pregnancy and social disadvantage: systematic review integrating controlled trials and qualitative studies. BMJ. 2009; 339: b4254. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBall M, Barnes J, Meadows P: Issues emerging from the first 10 pilot sites implementing the Nurse-Family Partnership home-visiting programme in England. London: Birkbeck, University of London. 2012; Reference Source\n\nOwen-Jones E, Bekkers M, Butler C, et al.: The effectiveness and cost-effectiveness of the Family Nurse Partnership home visiting programme for first time teenage mothers in England: a protocol for the Building Blocks randomised controlled trial. BMC Pediatr. 2013; 13(1): 114. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRobling M, Bekkers M, Bell K, et al.: Effectiveness of a nurse-led intensive home-visitation programme for first-time teenage mothers (Building Blocks): a pragmatic randomised controlled trial. Lancet. 2015. Publisher Full Text\n\nLugg-Widger FV, Cannings-John R, Channon S, et al.: Assessing the medium-term impact of a home-visiting programme on child maltreatment in England: protocol for a routine data linkage study. BMJ Open. 2017; 7(7): e015728. 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PubMed Abstract \| Publisher Full Text\n\nRyan M, Gerard K, Amaya-Amaya M: Using discrete choice experiments to value health and health care. 1 ed: Springer Netherlands; 2008. XX, 256. Reference Source\n\nLancsar E, Louviere J: Conducting discrete choice experiments to inform healthcare decision making: a user’s guide. Pharmacoeconomics. 2008; 26(8): 661–77. PubMed Abstract \| Publisher Full Text\n\nRyan M, Netten A, Skåtun D, et al.: Using discrete choice experiments to estimate a preference-based measure of outcome--An application to social care for older people. J Health Econ. 2006; 25(5): 927–44. PubMed Abstract \| Publisher Full Text\n\nRyan M, Bate A, Eastmond CJ, et al.: Use of discrete choice experiments to elicit preferences. Qual Health Care. 2001; 10 Suppl 1(Suppl 1): i55–i60. PubMed Abstract \| Free Full Text\n\nRyan M, Scott DA, Reeves C, et al.: Eliciting public preferences for healthcare: a systematic review of techniques. Health Technol Assess. 2001; 5(5): 1–186. PubMed Abstract \| Publisher Full Text\n\nEuroQol Group: EuroQol--a new facility for the measurement of health-related quality of life. Health Policy. 1990; 16(3): 199–208. PubMed Abstract \| Publisher Full Text\n\nBryan S, Roberts T, Heginbotham C, et al.: QALY-maximisation and public preferences: results from a general population survey. Health Econ. 2002; 11(8): 679–93. PubMed Abstract \| Publisher Full Text\n\nDrummond M, Sculpher M, Torrance G, et al.: Methods for the economic evaluation of health care programmes. Oxford, UK: Oxford University Press; 3rd edition, 2005. Reference Source\n\nWitt J, Scott A, Osborne RH: Designing choice experiments with many attributes. An application to setting priorities for orthopaedic waiting lists. Health Econ. 2009; 18(6): 681–96. PubMed Abstract \| Publisher Full Text\n\nBurge P, Netten A, Gallo F: Estimating the value of social care. J Health Econ. 2010; 29(6): 883–94. PubMed Abstract \| Publisher Full Text\n\nKuhfeld WF: Marketing research methods in SAS: Experimental design, choice, conjoint and graphical techniques. SAS Institute Inc, Cary, NC, USA, 2010. Reference Source\n\nStamuli E: Designs AD. Harvard Dataverse, V1, 2020. http://www.doi.org/10.7910/DVN/GK4TPV\n\nOrme B: Sample size issues for conjoint analysis studies. Sequim: Sawtooth Software Technical Paper, 1998. Reference Source\n\nStamuli E: Patient information sheet. Harvard Dataverse, V1, 2020. http://www.doi.org/10.7910/DVN/JBVRTZ\n\nOffice for National Statistics: Census, key statistics and quick statistics for local authoritis in the United Kingdom. 2011. Reference Source\n\nSurvey) NNR. Lifestyle data. 2012-13. Reference Source\n\nReed Johnson F, Lancsar E, Marshall D, et al.: Constructing Experimental Designs for Discrete-Choice Experiments: Report of the ISPOR Conjoint Analysis Experimental Design Good Research Practices Task Force. Value Health. 2013; 16(1): 3–13. PubMed Abstract \| Publisher Full Text\n\nBrazier J, Akehurst R, Brennan A, et al.: Should patients have a greater role in valuing health states? Appl Health Econ Health Policy. 2005; 4(4): 201–8. PubMed Abstract \| Publisher Full Text\n\nOlds DL, Henderson CR Jr, Chamberlin R, et al.: Preventing child abuse and neglect: a randomized trial of nurse home visitation. Pediatrics. 1986; 78(1): 65–78. PubMed Abstract\n\nKitzman H, Olds DL, Henderson CR Jr, et al.: Effect of prenatal and infancy home visitation by nurses on pregnancy outcomes, childhood injuries, and repeated childbearing. A randomized controlled trial JAMA. 1997; 278(8): 644–52. PubMed Abstract \| Publisher Full Text\n\nOlds DL, Robinson J, O'Brien R, et al.: Home visiting by paraprofessionals and by nurses: a randomized, controlled trial. Pediatrics. 2002; 110(3): 486–96. Publisher Full Text\n\nBarlow J, Barnes J, Sylva K, et al.: Questioning the outcome of the Building Blocks trial. Lancet. 2016; 387(10028): 1615–6. PubMed Abstract \| Publisher Full Text\n\nRobling M, Torgerson D, Pickett K, et al.: Questioning the outcome of the Building Blocks trial - Authors' reply. Lancet. 2016; 387(10028): 1616–7. PubMed Abstract \| Publisher Full Text\n\nOlds D: Building evidence to improve maternal and child health. Lancet. 2016; 387(10014): 105–7. PubMed Abstract \| Publisher Full Text\n\nDevelopments: New programmes evolving from the Family Nurse Partnership. 2017. Reference Source\n\nStamuli E: FNP Discrete Choice experiment. Harvard Dataverse, V2, UNF 6:/Cuxds1d8w7ucut5cyCNkQ== [fileUNF], 2020. http://www.doi.org/10.7910/DVN/DUL9KT\n\nStamuli E: Variables Key. Harvard Dataverse, V1, 2020. http://www.doi.org/10.7910/DVN/L8QOKC" }	[ { "id": "127314", "date": "04 Apr 2022", "name": "Liz Morrell", "expertise": [ "Reviewer Expertise Mixed methods exploration of preferences", "including discrete choice experiments" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 paper describing a discrete choice experiment among the UK general public. The study examines public preferences for the outcomes of a complex health and social care intervention designed to improve outcomes for young mothers and their children: the Family Nurse Programme, which has been evaluated in a randomised controlled trial. Given the large number of outcomes evaluated in the trial, this DCE aims to assess the relative value of those outcomes, to aid decision-making on the future of the FNP.\nThe work is largely clearly presented. There were a few areas that I suggest would benefit from additional information or clearer presentation:\nRespondent characteristics: it would be helpful to provide a national benchmark for educational achievement. I have used online providers for DCEs, and typically find over-representations of respondents with higher educational achievement; this is also reported in other published DCE's from UK academic researchers. I've used 2011 Census, although the categories might not line up exactly with the classifications presented - nonetheless, I feel it would be a useful addition. (In passing, note typo in ref 31 'authorities').\n\nSubgroup analyses: I found these paragraphs unclear. The paragraph on gender I think captures the interpretation clearly; however the subsequent paragraphs are less clear, implying that some subgroups derive satisfaction from negative outcomes for children or their mothers. Whilst this might make sense to economists used to thinking about utility, the interpretation may not be so obvious to a broader audience. Descriptions like 'derive higher disutility...' or even 'were less likely to choose...' are more intuitive. The final sentence in the parents/guardian paragraph is missing the comparator - they derived higher utility than who?\n\nTable 4: again for appeal to a broader audience, I would suggest defining or explaining some of the terms - for example the interpretation of the constants, sigma, rho. It would also help to explain numbers of observations and cases - I couldn't reconcile them with the sample size numbers provided, so an explanation would add clarity. I'm also not sure how to interpret the bold/not bold font.\n\nTable 5: could you note which is the reference category in the interaction? It's very difficult to interpret without knowing that. I assume the age interaction is binary (<=65, 65+)? - it would help to state that. It might help to break up the table with a sub-heading indicating where the interactions start, to direct the reader's eye. I'm also not sure how to interpret the colours of font - is there a meaning, or just to assist readability?\n\nDiscussion, paragraph on uptake: I found this unclear on first reading. As written I initially thought it was uptake by the young mothers, which would require a 'neither' option in the DCE. On re-reading, I realise it's about continued funding. Could that be clearer?\nA DCE is is an appropriate method, as it aims specifically to measure the relative value of the attributes presented. The qualitative piloting of the survey tool is good practice. I would, though, find it helpful to see an example of a choice-set, to get a clearer sense of precisely what the respondents were presented with and how the question was phrased. A limitation of the study that is not discussed is the selection of 10 secondary outcomes from a total of over 60, by an expert elicitation process; clearly choices had to be made and the approach is not unreasonable, but if an important attribute has been omitted that is a source of bias, and should be mentioned (perhaps in the Discussion, where the issue of omitted interactions is raised?)\nThe Methods section is largely clear, provides rationales for the choices made, and refers to general guidance for conducting DCEs. To allow understandability and ease of reading among a broader audience, I thought the paragraph on D-efficiency could include a layperson's explanation of D-efficiency and why it's important. For the same reason, the introduction of utility in the model section appears rather sudden - perhaps would benefit from a sentence explaining utility and its relevance to choices made in this type of study; this might also help understanding of the interpretations of the coefficients in the results section.\n\nReaders might be interested to know why one block had an additional choice-set. I would also like to know which analyses were pre-specified, and what other analyses were done but are not presented (interactions with other respondent characteristics?).\nThe random effects probit model is an appropriate statistical analysis. I have not done a blocked design of this type, so I am not able to comment on the statistical analysis of the blocking.\nThe relevant source data are provided.\nAs a non-expert in this field, I'm not convinced by the statement that the public value outcomes for the child above those for the mother - smoking, health status, and relationship with partner also have high coefficients. This is addressed in the first paragraph of the Discussion, but should perhaps be addressed in the Results. I accept there may be some aspect of how we assign these attributes as benefitting the mother or child that I'm missing here (I don't know this field at all) .\n\nI found the Conclusions paragraph a little confusing - it appeared to open up new issues about short-term effect measures and inter-pregnancy interval, which was interesting but I'm not sure added to my understanding. A shorter, clear statement of what the DCE contributes, might be a stronger Conclusion\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": [] }, { "id": "154354", "date": "23 Nov 2022", "name": "Anna Price", "expertise": [ "Reviewer Expertise Health services research", "randomised controlled trials", "first 1000 days", "early years" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 paper, which explores an interesting idea. My relevant experience for reviewing this paper is in nurse home visiting programs and community-based randomized trials, and not in DCEs or cost utility analyses. I agree with the comments raised by the first Reviewer. In addition, I am curious to understand the following:\nThe authors have justified the importance of eliciting preferences of the general public. However, if this is about consumer preferences, why wasn’t the research run with the subpopulation who is likely to be eligible for the program, which is likely to be distinct from the general population? This is noted in the Discussion (with a sentence I think could be strengthened to advocate more for specific consumer involvement) and I think the paper would be strengthened with a justification in the Introduction as well.\n\nWith regards to the description of the cohort as broadly representative, I am not sure I agree with this apart from the age distribution. What are the implications of these cohort differences on the study findings? I think the paper could be strengthened with a tempering of the wording regarding representativeness, and a discussion of the potential limitations of the sampling distribution. Like Reviewer 1, I think it’s important to include education data where possible.\n\nAs a non-economist, I found it difficult to understand the Results sections and match the text with the Tables, particularly with the wording around utility and disutility. For example, I don’t understand the following: “the negative signs of the coefficients would suggest that respondents’ utility decreases as the level of the attribute increases, or becomes a negative outcome (0= positive outcome, 1=negative outcome). Positive signs would indicate the contrary i.e. respondents derive higher utility as the level of the attribute decreases (i.e. becomes a “positive outcome”).” Is it possible to illustrate what this means with examples from the Table? As such, I have not reviewed the Methods or the interpretation of the Results and recommend a Specialist is engaged to review these sections.\nWith thanks and best wishes, Anna\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?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-677
https://f1000research.com/articles/9-676/v1	03 Jul 20	{ "type": "Brief Report", "title": "An equation for the biological transmembrane potential from basic biophysical principles", "authors": [ "Marco Arieli Herrera-Valdez" ], "abstract": "Biological membranes mediate different physiological processes necessary for life, many of which depend on ion movement. In turn, the difference between the electrical potentials around a biological membrane, called transmembrane potential, or membrane potential for short, is one of the key biophysical variables affecting ion movement. Most of the existing equations that describe the change in membrane potential are based on analogies with resistive-capacitive electrical circuits. These equivalent circuit models assume resistance and capacitance as measures of the permeable and the impermeable properties of the membrane, respectively. These models have increased our understanding of bioelectricity, and were particularly useful at times when the basic structure, biochemistry, and biophysics of biological membrane systems were not well known. However, the parts in the ohmic circuits from which equations are derived, are not quite like the biological elements present in the spaces around and within biological membranes. Using current, basic knowledge about the structure, biophysics, and biochemical properties of biological membrane systems, it is shown here that it is possible to derive a simple equation for the transmembrane potential. Of note, the resulting equation is not based on electrical circuit analogies. Nevertheless, the classical model for the membrane potential based on an equivalent RC-circuit is recovered as a particular case, thus providing a mathematical justification for the classical models. Examples are presented showing the effects of the voltage dependence of charge aggregation around the membrane, on the timing and shape of neuronal action potentials.", "keywords": [ "Transmembrane potential", "membrane biophysics", "membrane physiology", "excitable membrane", "computational neuroscience", "computational physiology." ], "content": "Introduction\n\nBiological membranes mediate communication between cellular compartments and their surrounding environments (Blaustein et al., 2004; Boron & Boulpaep, 2016; Helman & Thompson, 1982; Sten-Knudsen, 2002). One of the most important biophysical variables affecting, and affected by such communication, is the difference between the electrical potentials inside and outside the cellular membrane, called transmembrane potential, or membrane potential, for short (Johnston et al., 1995; Sperelakis, 2012; Weiss, 1996). Paraphrasing Cole (Cole, 1933), the existence of the membrane potential is, by itself, an indication of the fact that a cell is alive. The ongoing changes in the membrane potential are physiological correlates of cellular activity in different levels of organization, within the cell itself and its surroundings. Therefore, it is important to have a basic understanding of the biophysical principles underlying its behaviour.\n\nThe transmembrane potential is generated by the presence of ions of different species on both sides of the membrane (Briggs, 1930; Brooks, 1929) and differences in their passive transmembrane permeabilities (Cole, 1940; Fricke, 1925; Höber, 1936). The membrane potential is also dynamically affected by passive and active transport processes (Blaustein et al., 2004; Boron & Boulpaep, 2016) that underlie electrical signaling and other physiological functions (Eisenberg, 1990; Eisenberg, 1998; Hille, 1992; Höber et al., 1939; Hodgkin & Katz, 1949a; Hodgkin & Katz, 1949b). Conversely, ion transport is itself dependent on changes in the transmembrane potential.\n\nModeling approaches have been useful to describe electrical and biochemical interactions involving ions, and how they affect the membrane potential (Fricke, 1925; Osterhout, 1933; Teorell, 1935). Many models of biological membranes are based on constructing equivalent electrical circuits (Hermann, 1899, Hermann, 1905) using conservative paradigms like Kirchoff’s law of electrical current for RC-circuits (Cole, 1933; Fricke, 1931; Hermann, 1872; Johnson et al., 1989). Of seminal importance, the Hodgkin & Huxley (1952) model describes the changes in membrane potential with a system of differential equations derived from an RC-circuit. The equations in the model are derived by considering an electrical circuit with resistors, a capacitor, and a battery arranged in parallel, respectively representing what is known today as membrane channels, the neighborhood around the membrane, and the membrane potential. The total current in the model is the sum of a “capacitative” current representing flow of electrical charge around the membrane, and different resistive currents representing different transmembrane ion fluxes, now known to be mediated by channels selective to ions of specific types (Hodgkin et al., 1952). Ionic transmembrane currents are, however, not resistive, as ions cross the membrane either by diffusion (Eisenberg, 1998; Neher & Sakmann, 1976), or via mechanical translocation by protein pumps (Gadsby, 2009). However, ions moving across a membrane are not like electrons through a wire and the lipid bilayer of the membrane is not quite like the air-filled space between the plates in a capacitor.\n\nIn partial agreement with the basic postulates of models based on equivalent circuits, present knowledge about the molecular structure and biochemistry of biological membranes supports the idea that charged molecules accumulate next to the hydrophilic heads of the lipid bilayer that forms the outer portion of the membrane, and that the density of charge around the membrane is increasing as a function of the membrane potential (Everitt & Haydon, 1968; Wobschall, 1972). One important assumption for the equivalent circuit, is that the charge aggregation around the membrane is proportional to voltage, which implies that the time-dependent rate of change of such a flow is assumed to be constant. Several studies report that the specific membrane capacitance for membranes of multiple cell types is approximately 1 µF/cm2 (Cole & Curtis, 1938a; Cole & Curtis, 1938b; Cole & Hodgkin, 1939; Galligan et al., 2000; Gentet et al., 2000). The charge density around the membrane is, however, not necessarily proportional to the membrane potential (Kilic & Lindau, 2001; Weinberg, 2015). One reason for the potential divergence is that there are multiple problems associated to measuring the membrane capacitance experimentally (Golowasch et al., 2009; Weinberg, 2015). Perhaps the most important issue in this regard is the non-isopotentiality of the electric fields around membranes.\n\nShown herein, it is possible to derive a simple differential equation that describes the change in the transmembrane potential in a small patch of membrane from basic biophysical considerations, without using electrical circuit analogies. Importantly, a particular case of this generalised formulation explains the phenomenological formulation based on an equivalent circuit originally proposed by Hermann, Hodgkin and Huxley, and others (Hille, 1992). The generalisation and the possibility of explaining an already existing model are in line with the thinking behind the thermodynamic model for transmembrane transport (Herrera-Valdez, 2018), which proposes a general formulation for the molecular fluxes mediated by different physiological transmembrane transport mechanisms, both passive and active, and explains conductance based models as linear approximations around the reversal potential in the case of electrogenic transport.\n\nOnce the derivation is explained, it is illustrated with two simple examples of membrane excitability.\n\n\nBiophysical derivation to describe the transmembrane potential\n\nConsider a small volume Ω containing a small portion of a cellular membrane, together with the extracellular and intracellular compartments in the immediate neighborhood around the membrane. Assume, each of these compartments is filled with physiological solution containing ions of different species, and assume also that the number of ions for each of the different ion species is constant. Let Qa represent the density of charge around the membrane (Coulombs/µm2). Assume also that each of the ion types has some permeability across the membrane, and let Qm represent the charge density in the space within the lipid bilayer of the membrane (Coulombs/µm2). By extension, the total number of ions in the system and the total charge density Qa + Qm (in Coulombs) are constant with respect to time.\n\nLet Ue(t) and Ui(t) (in Volts) represent the electrical potential in the extra- and intracellular compartments, respectively. The difference v(t) = Ui(t) − Ue(t) is the transmembrane potential, or by simplicity, membrane potential. For conceptual simplification, if the extracellular potential is equal to zero, then the transmembrane potential could be thought of as the intracellular potential. Since ions tend to accumulate around the membrane as v increases in size, whether positive or negative, the net charge density (Coulombs/µm2) around the membrane can be assumed to be some smooth, monotonic, increasing function Qa(v), equal to zero for v = 0 (Figure 1A).\n\n(A) Three profiles for Qa(v), the voltage-dependence of the charge density around the membrane. The three functions can be regarded as similar around v = 0 and (B) Rate of change for the three Qa profiles as a function of voltage.\n\nIt follows from the assumption of constant total charge that the time-dependent change in the total charge density is zero. That is,\n\nHere ∂t represents the instantaneous rate of change with respect to time. Therefore, the term ∂tQm(t) represents the current density (Amperes/µm2) carried by different ions across the membrane and ∂tQa(v(t)) represents the current density around the membrane.\n\nEquation (1) yields a differential equation for v in terms of the ionic flux across the membrane, normalized by rate of change in charge density around the membrane with respect to v. To see this, note first that applying the chain rule to the term representing the charge density around the membrane yields\n\n\n\nwith ∂tv in V/s and ∂vQa in Coulombs/µm2 per Volt. Also, the current density across the membrane can be thought of as proportional to the total transmembrane flux of charge. Substitution of Equation (2) into Equation (1), and rewriting after solving for ∂tv yields\n\nThe multiplicative inverse of ∂vQa can be thought of as a scaling factor for the change in the membrane potential. Note Qa is cannot be a constant. If Qa is a linear function of v, then the scaling is a constant. If Qa is nonlinear as a function of v, then the scaling changes dynamically with v, possibly amplifying or dampening, depending on the way v changes in time. These possibilities are explored by analysing the effects of the profile of charge accumulation around the membrane on the generation of neuronal action potentials.\n\nAn important particular case that gives theoretical justification to the equivalent circuit analogy for membranes as a linear approximation is analysed in the following section. Having done that, the behaviour of the dynamics for v are explored using two nonlinear profiles for Qa (Figure 1A) that behave similarly to the linear case near v = 0, but differ in that one saturates, and the other grows exponentially, as the membrane is polarised.\n\nAssume that charge aggregation around the membrane is linear as a function of voltage. That is,\n\nThe change in the density of charge around the membrane, ∂vQa, is in Coulombs per volt per µm2, which is equivalent to farads/µm2, the units used for electrical capacitance. The plates of a capacitor in an electrical circuit are made of conducting media (e.g. metal), and separated by air, or other insulating material. Therefore, this particular case supports the idea that the intra and extracellular media can be thought of as similar to the metal plates in the capacitor, and the membrane can be regarded as analogue to the insulating air layer between the plates.\n\nIt is arguable that there is a voltage-dependent upper bound for the accumulation of charges around the membrane (Figure 1A, blue curve). That is, if the membrane is polarised to very large voltages (either negative or positive) the charge density around the membrane could be assumed to reach a limit. One possible description for the voltage-dependence of Qa could be\n\nAnother possibility similar to the current densities from the thermodynamic model (Herrera-Valdez, 2018), is that Qa(v) is a hyperbolic sine. In this case\n\nThe graph of Qa(v) is odd and increasing with exponential growth in both directions of the v axis (Figure 1A, orange curve), qualitatively opposite to that described earlier for the saturating Qa with hyperbolic tangent shape. In this case ∂vQa has a \"U\" shape with a local minimum value of Cm at v = 0, which means that ∂vQa exerts an attenuation effect on ∂tv that increases as v is further from 0 (Figure 1B, orange curve).\n\nNote the three profiles for the charge density around the membrane presented above can be thought of as approximations of one another around v = 0.\n\nHow does the voltage-dependence in the charge-density around the membrane influence the changes in the transmembrane potential? Specifically, what are the effects of the nonlinearities in the voltage-dependence of ∂vQa on the dynamics of the transmembrane potential? To compare the effects caused by the three profiles (tanh, linear, and sinh) for Qa, the dynamics for the transmembrane potential in a neuron were modelled (Figure 2, blue, black, and orange lines, respectively).\n\nThe panels on the left column (A,C,E) show time series for v, Qa, and 1/∂vQa, respectively. The blue, black and orange lines correspond to the saturating, linear, and exponential profiles for Qa, respectively. The panels on the right columns (B,D,F) show the corresponding trajectories of the same variables as a function of ∂tv, the instantaneous change in v with respect to time.\n\nConsider a neuronal membrane and assume that it has three transmembrane transport mechanisms, say, voltage-dependent K+ and Na+ channels, and a Na+-K+ ATPase. If w represents the proportion of open K+ channels and the proportion of inactivated Na+ channels, then the dynamics of the membrane potential can then be written as\n\nA description of all the parameters and their values can be found in Table 1.\n\nThe reversal potential for the Na+-K+ATPase is given by vNaK = vATP + 3vNa –2vK. Initial conditions at v0 = −48 mV and w0 = 0.001. Note 1µF/cm2 = 10−2 pF/µm2.\n\nNote that the ranges for the three different profiles for ∂vQa (Figure 1B) can be ordered by magnitude. The hyperbolic profile for Qa yields the lowest values for ∂vQa, in comparison to the constant ∂vQa = Cm, and the hyperbolic sine profile, which yields the largest values for ∂vQa. Therefore, the scaling effects of 1/∂tQa on the change in transmembrane potential can be thought of in the opposite order, with larger effects caused by saturating Qa, a smaller scaling for the linear profile, and the smallest scaling effect caused by the exponential profile. It is possible to appreciate this by comparing the trajectories of three neuronal action potentials from the same initial conditions (v0 = −48 mV and w0=0.001), obtained from the model in equations Equation (9)–Equation (16) using the same parameters (Herrera-Valdez et al., 2013), and the same initial conditions, except for their Qa profile (Figure 2).\n\nAs anticipated, the smallest scaling effect on the change in v occurs for the hyperbolic sine profile (Figure 2, orange curves, spike time at 6.62 milliseconds approx), as shown by the longer delay in the action potential in comparison with the linear and saturating profiles (Figure 2A–B, black and blue curves, with spike times at 13.63 and 20.82 milliseconds, respectively). The delays to the action potential can be increased if the initial condition is lower, or decreased if higher, without altering the hierarchy in the scaling effect.\n\nThe scaling effects on ∂tv caused by the three Qa profiles can also be appreciated in the trajectories of the (∂tv,v)-plane, which shows a smaller area contained by the curve for the exponential-profile, in comparison to the areas bounded by trajectories produced by the linear and saturating profiles for Qa, respectively.\n\nThe dynamical ranges for Qa for the different profiles show smaller departures from 0 in the saturating profile with respect to the other two profiles (Figure 2C–D). This suggests that that measuring charge aggregation around the membrane during action potentials would not necessarily show large differences indicative of the Qa profile, at least with the assumptions made here.\n\nIn contrast, the scaling induced by the inverse of ∂vQa is notorious in both its time-dependence of the scaling factor, and also in its trajectory as a function of ∂tv (Figure 2E–F). The larger scaling effect of the saturating profile on ∂tv can be clearly appreciated in Figure 2F, which shows the values of the scaling factors of the saturating profile during the action potential above any of the values from the linear or exponential profiles.\n\nOne indirect way of measuring the effects of the scaling factor 1/∂tQa on the change in membrane potential, ∂tv, is to calculate the efficiency of the Na+current during the upstroke of the action potential (Carter & Bean, 2009). This is done by calculating the total Na+charge during the upstroke, and dividing it by the total charge during the same time interval. Explicitly, if starting from an initial condition v = v0, the time interval between the start of the trajectory and the peak time of an action potential is [0, T]\n\nScaling factors with more dampening effects would cause the Na+current to be less efficient.\n\nTo further illustrate the scaling effects of the Qa profiles on ∂tv, the maximum upstroke velocities, the time delays to produce action potentials, and the efficiency of the Na+current during the upstroke were calculated from sample trajectories obtained by increasing the values of the initial condition for v (Table (2)). In agreement with the observations made above, the decreasing amplification caused by the saturating and linear profiles (in that order) with respect to the exponential profile were negatively correlated with the efficiency, and the delay to spiking. Nevertheless, it is worth noticing that the differences decrease for larger values of v0.\n\n\nDiscussion\n\nA simple equation describing the time evolution of the transmembrane potential has been derived from basic biophysical principles (Equation (3)). The new equation is in line with the derivation of the thermodynamic model (Herrera-Valdez, 2018), in that it only considers basic biophysical principles to describe the elements in a system that include a membrane, and ions in solution. Importantly, the derivation is not based on an equivalent circuit analogy, but it is general enough to allow the possibility of obtaining the traditional equation for the equivalent RC-circuit. The derivation draws from basic, current knowledge about the membrane and its surroundings and separately taking into account its ion-impermeable and ion-permeable properties, as referred by early works by Cole and Curtis and other authors. As it is desirable in any generalization, it explains a classical model proposed in seminal work previously done. Explicitly, the expression for the change in voltage-dependent in the charge density around the membrane can be simplified to obtain the so-called “membrane capacitance” in classical models. This is done by assuming that the charge density around the membrane is linearly related to the membrane potential. Interestingly, the term emerges from applying the chain-rule to calculate the time-dependent change in the total charge density around the membrane, which depends on voltage (Coombs et al., 1959; Everitt & Haydon, 1968; Lu et al., 1995).\n\nFrom a physical perspective, changes in the sign and magnitude of the transmembrane potential should cause the charges around the membrane to redistribute (Huang, 1981; Santos-Sacchi & Navarrete, 2002). Such changes occur within very short times relative to the usual time scales taken into account for studies of membrane excitability, or electrical signalling in general. One important result from the derivation is that the change in the charge density around the membrane is not necessarily a constant, as evidenced by different reports (Amzica & Neckelmann, 1999; Neher & Marty, 1982; Proks & Ashcroft, 1995; Smith et al., 1997).\n\nFor historical interest, prior to 1938 approximately, the hypothesis the membrane would disintegrate at the pass of the excitation wave during an action potential was a source of debate and reason for experimentation (Cole & Curtis, 1938a). The model presented here provides a theoretical argument that explains how changes in ∂vQa can take place without membrane disintegration, and enables the possibility of studying membrane systems without assuming that the amount of charge aggregation around the membrane is proportional to voltage (Everitt & Haydon, 1968; Kilic & Lindau, 2001). Cole and Curtis dismissed that hypothesis on the basis of finding very small changes in the membrane capacitance using a Wheatstone bridge to record currents from squid axons and other preparations. Of note, the change in accumulation of charge around the membrane, commonly called \"membrane capacitance\" is typically regarded as a constant since around the time of the seminal work of Cole in the decade of 1930’s (Cole & Curtis, 1938b; Cole & Curtis, 1939), and others (Everitt & Haydon, 1968). Further, more recent studies have also found non significant changes in membrane capacitance (Gentet et al., 2000). One problem is that such measurements have been made for membrane potentials around rest. From a theoretical perspective, one reason that experimental measures of membrane capacity have proved to be difficult, is the assumption of isopotentiality, which does not hold almost surely (Chen & Gillis, 2000; Golowasch et al., 2009). Nevertheless, from a physical perspective, the change in the charge density with respect to voltage, ∂vQa, can be a non-constant function (Santos-Sacchi & Navarrete, 2002). Indeed, it has been shown in experimental work involving exo and endocytosis (Neher & Marty, 1982; Proks & Ashcroft, 1995; Smith et al., 1997), and in relation to voltage-gated currents (Kilic & Lindau, 2001). The profiles for saturation and exponential aggregation of charge around the membrane defined above suggest that experimental measures of the impermeable aspect of the membrane as originally called by Cole and Curtis and others, may be even more difficult than previously thought (but see (Kilic & Lindau, 2001)).\n\nOf note, the runs with the saturating Qa profile suggest a reasonable explanation for the rapid changes during the initiation of an action potential (Naundorf et al., 2006), as they suggest a way to accelerate the initial depolarisation during the action potential upstroke that cannot be obtained from the Hodgkin and Huxley model.\n\nOne issue of possible importance is the timing of action potentials in a network context, which, on top of being influenced by local field potential oscillations (time-dependent forcing on Equation (3), could be subject of non-negligible rescaling effects due to voltage-dependent changes in ∂vQa (Amzica & Neckelmann, 1999). Future experimental measurements may shed light on this issue, as it could have large repercussions in our interpretations of network dynamics.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Acknowledgements\n\nA previous version of this article is availiable on BioRxiv: https://doi.org/10.1101/2020.05.02.073635.\n\n\nReferences\n\nAmzica F, Neckelmann D: Membrane capacitance of cortical neurons and glia during sleep oscillations and spike-wave seizures. J Neurophysiol. 1999; 82(5): 2731–2746. PubMed Abstract \| Publisher Full Text\n\nBlaustein MP, Kao JPY, Matteson DR: Cellular physiology. Elsevier/Mosby, ISBN 0323013414, 2004. Reference Source\n\nBoron WF, Boulpaep EL: Medical Physiology E-Book. Elsevier Health Sciences. 2016. Reference Source\n\nBriggs GE: The accumulation of electrolytes in plant cells—a suggested mechanism. Proceedings of the Royal Society of London. 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Publisher Full Text" }	[ { "id": "74256", "date": "26 Nov 2020", "name": "James Weifu Lee", "expertise": [ "Reviewer Expertise Protonic bioenergetics and membrane potentials" ], "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\nReview Comments This is a quite interesting manuscript, in which, the author tries “to derive a simple equation for the transmembrane potential”. As Dr. Herrera-Valdez indicated, the biophysical origin of membrane potential was indeed not entirely clear in the past 50 years until very recently. Discussions and research efforts on bioenergetics including membrane potential should be encouraged. This reviewer likes Dr. Herrera-Valdez’s sprits of out-of-box thinking. However, some of the assumptions in this manuscript seem quite questionable. There are spots that need major improvements before this manuscript could be considered for indexing.\nFirst, Dr. Herrera-Valdez seems not aware of the latest development in the field of protonic bioenergetics and membrane potential. Actually, there is already an unified framework of protonic bioenergetics with a newly formulated membrane potential equation in resolving the issues of the Mitchellian chemiosmosis: a new transmembrane electrostatic proton localization theory that has recently resulted in a new protonic action potential equation (2020 Journal of Neurophysiology, 124 (4): 1029–1044; 2020 Scientific Reports, 10:10304; 2020 ACS journal Omega 5 (28): 17385-17395; Heliyon 4 (2019) e01961; 2015 Bioenergetics 4: 121. doi:10.4172/2167-7662.10001211).\nThis reviewer encourages the author to consider the latest development including the latest membrane potential equation from the transmembrane electrostatic proton localization theory and build a better case for the science of membrane potential.\nThe manuscript says “let Qm represent the charge density in the space within the lipid bilayer of the membrane (Coulombs/μm2)”. This assumption seems quite questionable or problematic. Is there any scientific evidence for the existence of charge density Qm “in the space within the lipid bilayer of the membrane”? Is the Qm referring to the charges of membrane phospholipid head groups or something else since the “space within the lipid bilayer of the membrane” is hydrophobic (essentially zero charges)?\nThe statements “Here ∂t represents the instantaneous rate of change with respect to time. Therefore, the term ∂tQm(t) represents the current density (Amperes/μm2) carried by different ions across the membrane and ∂tQa(v(t)) represents the current density around the membrane” need to better be explained and justified. For example, does the ∂tQa(v(t)) represents the current density in the bulk liquid phase around the membrane or not?\nAuthor’s Eq. 3 seems missing a negative sign.\nThe manuscript says: “if the membrane is polarised to very large voltages (either negative or positive) the charge density around the membrane could be assumed to reach a limit”. Is there any evidence for such a case? In reality, there is no evidence for such a case in any biological systems. But, the author seems arbitrarily use a hyperbolic tangent \"tanh\" function to describe the voltage-dependence of Qa (Eq. 5) without justification.\nThe manuscript also says: “Another possibility similar to the current densities from the thermodynamic model (Herrera-Valdez, 2018), is that Qa(v) is a hyperbolic sine”. Is there any experimental evidence or justification for this case?\nSo, the manuscript leaves with three cases: linear, hyperbolic tangent \"tanh\", and hyperbolic sine \"sinh\"; Which one you want people to believe?\n\nIt is questionable whether a paper like that would cause confusion in the field.\nTherefore, if the author could objectively address these important issues, this reviewer would consider support the indexing of his revised manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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? No\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": "127237", "date": "28 Mar 2022", "name": "Christian Brosseau", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI do not recommend the indexing of this manuscript as is because it does not add any substantial physical content to the existing literature. That is not applied physics at the level we expect. This manuscript appears to contain a presentation of data without the in-depth analysis expected. Hence, it is not sufficient to report the outcomes of experiments without drawing out new physics in the interpretation of the results. In this manuscript, there are descriptions of the Figures, but there is little said about their physical significance and what new understanding can be gained from them.\nIn addition to not satisfying this primary requirement, there are a number of other problems with the manuscript which together make it unsuitable for further review. For example, the English language is not of the standard required for indexing.\nThe title is misleading: \"biological transmembrane potential\" should be changed to \"cell transmembrane potential\".\nSeveral important references dealing with the subject at hand are missing and are eventually not discussed, e.g. \"Resistor-capacitor modelling of the cell membrane: a multiphysics analysis\", J. Appl. Phys., 129, 011101 (2021).1\nThe author should precise the exogeneous voltage excitation and how the temporal variation of the potential can impact the membrane (and cell) parameters which eventually regulate the overall electromechanical behavior of the cell.\nSeveral equations are not based on first principles of physics or result from an educated guess which are not fully substantiated, e.g. Eq. (7); what are the assumptions and limitations associated with this equation? Unclear.\nSeveral statements are also unclearly discussed, e.g. “Consider a neuronal membrane and assume that it has three transmembrane transport mechanisms, say, voltage-dependent K+ and Na+ channels, and a Na+-K+ ATPase.\" How are the ionic channels are considered here?\nSince the author does not explain what is the physics associated with the data, how all of this can be actually measured and checked, Fig. 2 is completely not understandable.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/9-676
https://f1000research.com/articles/9-675/v1	03 Jul 20	{ "type": "Review", "title": "Post-traumatic stress in the wake of the COVID-19 pandemic: a scoping review", "authors": [ "Ravi Philip Rajkumar" ], "abstract": "Background: Post-traumatic stress symptoms (PTSS), as well as post-traumatic stress disorder (PTSD) itself, are common sequelae of disasters, including disease outbreaks such as the COVID-19 pandemic. Understanding their frequency and correlates is essential to developing preventive, therapeutic and supportive measures in a time of crisis. Methods: A scoping review of the literature pertaining to PTSS and PTSD in relation to COVID-19 was carried out with the primary objective of assessing the frequency of PTSS/PTSD and the factors associated with it, and the secondary objective of summarizing expert recommendations in this field. For this purpose, PubMed and Embase literature databases were searched using the terms “COVID-19”, “nCoV-2”, “post-traumatic stress disorder”, “PTSS”, “PTSD” and “traumatization” in various combinations. A total of 10 relevant publications were found, which were tabulated and organized into original research on PTSS/PTSD (n = 7) and expert opinions or reviews (n = 3). Results: The frequency of PTSS ranged from 7-34%, depending on study population and methodology. Gender, marital status, physical symptoms, and a prior psychiatric diagnosis were associated with the severity of PTSS. Expert opinions highlighted the prolonged nature of the impact of COVID-19, the need for long-term preventive and treatment strategies, and the need for innovation and collaboration in research and dissemination of information. Conclusions: The COVID-19 outbreak is likely to leave a large number of persons suffering from PTSD in its wake. The above results may help us to identify those at risk in order to deliver preventive or early therapeutic interventions.", "keywords": [ "post-traumatic stress disorder", "vicarious traumatization", "COVID-19" ], "content": "Introduction\n\nLarge-scale disasters, such as disease outbreaks, are associated with an increased risk of post-traumatic stress symptoms (PTSS) or post-traumatic stress disorder (PTSS), both in the general population and in high-risk groups such as patients and healthcare workers. Research from earlier outbreaks of a novel coronavirus disease has shown that 25.6% of survivors of the severe acute respiratory syndrome (SARS) outbreak fulfilled criteria for PTSD at 30 months’ follow-up1; similarly, both healthcare workers and patients under quarantine reported high levels of PTSS during the Middle East respiratory syndrome (MERS) outbreak2. Compared to these earlier outbreaks, which were limited in geographical scope, the global outbreak of novel coronavirus disease (COVID-19) has affected nations all across the globe, with over 3.6 million cases reported to date; therefore, it is logical that this pandemic would have a more profound and widespread psychological impact than its predecessors3,4. Some authors have already raised the concern of a “tsunami” of post-traumatic stress disorder sweeping across the globe in the wake of the COVID-19 crisis5, an image which vividly captures the scope of this impact.\n\nThe factors involved in psychological traumatization and the development of PTSD during the COVID-19 outbreak are complex and interlinked. To the direct effects of the disease and the fear of infection and death must be added the widespread hypervigilance that is often encouraged by authorities6, limitations in the availability of healthcare and other resources5, the traumatic effects of measures such as lockdowns, curfews and quarantine7, experiences of stigma and discrimination8, social isolation and financial hardship9 and misinformation about the disease and its origins4. Moreover, prolonged contact with those who have been traumatized can itself be traumatic to some, a phenomenon known as vicarious traumatization10. Certain groups of people have been identified as being more vulnerable to traumatization during the COVID-19 outbreak, including healthcare professionals and other front-line workers11, children and adolescents12, the elderly13, and those with pre-existing psychiatric disorders14. More than one of these risk factors or mechanisms may be operative in a given individual.\n\nThus, the following review was carried out to obtain a preliminary estimate of (a) the frequency of PTSD or PTSS in various populations exposed to the COVID-19 pandemic, and (b) the factors identified as having an association with PTSS and PTSD in published reports. As a secondary objective, a review of expert opinions and commentaries published in this area was also undertaken.\n\n\nMethods\n\nThe current paper is a scoping review of literature related to PTSD/PTSS during the COVID-19 outbreak. The review protocol for the same is described below, and it was carried out in accordance with the PRISMA-Scoping Review extension guidelines15. It has not been formally registered, and further details are available on request from the author.\n\nAll articles which provided either an estimate of the frequency of PTSS or PTSD in relation to the COVID-19 pandemic, the factors associated with such symptomatology, or expert opinions regarding research or patient care in this area, published in the English language between January 1, 2020 and May 15, 2020, were considered for inclusion in the study. The most recent search was conducted on May 17, 2020. Articles were retrieved through a search of the PubMed and Embase databases using the terms “COVID-19”, “nCoV-2”, “post-traumatic stress disorder”, “PTSS”, “PTSD” and “traumatization” in various combinations. For example, for the PubMed database, the search strategy was as follows:\n\n1. “COVID-19” (Medical Subheading, Title, Abstract, Text)\n\n2. “Severe Acute Respiratory Syndrome Coronavirus 2” (Supplementary concept)\n\n3. “Post-traumatic Stress Disorder” (Medical Subheading, Title, Abstract, Text)\n\n4. “Post-traumatic Stress” OR “PTSS” OR “PTSD” OR “traumatization” (Title, Abstract, Text)\n\n5. 1 AND (3 OR 4)\n\n6. 2 AND (3 OR 4)\n\nA total of 36 citations were retrieved from PubMed and 38 from EMBASE. After discarding duplicate citations, a total of 36 citations were screened according to the eligibility criteria mentioned above. 12 articles were excluded at this stage as they dealt with topics unrelated to PTSD / PTSS, four were excluded because they were general commentaries on PTSD (without observational data or recommendations) published in Chinese, French or German, and ten were excluded because they were general commentaries on public or mental health that did not specifically discuss PTSD. A total of ten papers – seven observational studies and three expert commentaries, were selected for charting and data synthesis. The details of this process are summarized in Figure 1.\n\nData was charted in two standardized forms by the author. The first form, which covered the seven observational studies, summarized the data under the following headings: (a) study location; (b) study population (for example: general public, youth, or healthcare workers); (c) psychometric instrument used to measure PTSS and cut-off value used by the authors; (d) reported frequency of PTSS / PTSD expressed as a percentage; (e) subgroup analysis, only if explicitly described by the original researchers (for example: healthcare workers versus the general public); and (f) variables that were significantly associated with an elevated or reduced risk of PTSS / PTSD, as assessed by the author after reviewing all published data (tables and text) provided by the original researchers. Owing to the small number of studies and the significant heterogeneity in study populations, methodology and outcome measures, a formal statistical summation, such as a pooled prevalence or confidence interval, was not computed. Instead, results were tabulated and presented below in two sections, the first providing estimates of frequency and the second providing details of statistically significant correlates.\n\nThe second form, which covered the three expert reviews and recommendations, summarized the data under the following headings: (a) recommendations for prevention of PTSS / PTSD; (b) recommendations for treatment of those identified as having such symptoms; (c) recommendations for policy; and (d) recommendations for future research. As this data was qualitative, no formal statistical analysis was carried out.\n\n\nResults\n\nDetails of the citations assessed, excluded and selected for inclusion in this review are summarized in the PRISMA flow diagram (Figure 1). Of a total of 36 citations screened for inclusion, 26 were excluded for the following reasons: unrelated topic (n = 12), language other than English (n = 4), and general review / commentary not specifically addressing PTSD / PTSS (n = 10). A total of ten papers – seven original research studies and three expert commentaries – were selected for the final review, and these are discussed below.\n\nFive studies reported frequencies and percentages of post-traumatic stress symptoms16–20. The results of these studies are summarized in Table 1. All these studies used screening instruments administered remotely through computers or smartphones, with a pre-defined cut-off to estimate the presence of significant PTSS; in none of them was a formal diagnosis of PTSD confirmed using standard criteria. The most frequently used instrument was the Impact of Event Scale – Revised (IES-R), with a cut-off of 24 used in two papers16,17 and 18 in one paper18. Estimates of PTSS ranged from 7–34%, with a pooled frequency of 307/2633 (11.66%) across all studies. The use of a lower cut-off value for the IES-R was associated with the highest reported rates of PTSS across these studies.\n\n† For the entire sample; details of frequencies in each sub-group are discussed in the text (Results, Factors associated with PTSS during the COVID-19 pandemic).\n\nPTSS, post-traumatic stress symptoms; PTSD, post-traumatic stress disorder; DSM-5, Diagnostic and Statistical Manual of Mental Disorders 5th edition.\n\nA longitudinal study of individuals from 190 Chinese cities measured various psychological outcomes, including PTSS, in the general public at two time points: the first during the initial COVID-19 outbreak (n = 1210), and the second, four weeks later, during the peak of the epidemic in China (n = 861)21. The mean IES-R score in this sample decreased significantly over a period of four weeks, from 32.98 to 30.76 (p < .01); however, even the follow-up score remained above the pre-defined threshold (24) for significant PTSS, indicating that these symptoms persisted at follow-up, albeit at somewhat lower levels.\n\nA study of vicarious traumatization in a Chinese population, involving 214 members of the general public and 526 nurses and using 38-item questionnaire, found higher levels of vicarious traumatization in the general public (p < .001) and non-front line nurses (p < .001) compared to nurses involved in the front-line care of COVID-19 patients22; however, PTSS were not directly measured in this study.\n\nA number of variables were associated with the presence and severity of PTSS as a result of the COVID-19 pandemic. Female gender was associated with the severity of PTSS in two studies19,20, but one paper found an association with male gender; however, this study was specifically focused on people returning to work during the pandemic16. Other socio-demographic predictors of significance included younger age (12–21 years), a higher number of persons per household21, widowed, separated or divorced marital status16,19, lower educational qualifications, and employment in the business sector18. Physical symptoms resembling those of COVID-19, such as fever, sore throat, cough, myalgia and fatigue, were associated with PTSS in two studies17,20, one of which involved health workers exclusively; other physical variables associated with PTSS were self-rated poor health16 and poor sleep quality20. Pre-existing anxiety disorder or depressive disorder were associated with a higher frequency of PTSS than that observed in controls without a psychiatric diagnosis (43.4% vs 27.5%; p = .025)17. Finally, individual and institutional adherence to hygienic precautions, such as hand hygiene and social distancing, were associated with reduced severity of PTSS16.\n\nIn a paper examining the methodological and practical gaps related to the assessment of PTSS during the COVID-19 pandemic, Horesh and Brown provided several relevant recommendations6. From a conceptual viewpoint, they highlighted the fact that trauma and PTSD were not yet part of the public discourse on the impact of COVID-19, as opposed to general terms such as “anxiety” and “fear”. From a methodological viewpoint, they discussed the potential usefulness of machine learning methods and ecological momentary assessments in studying PTSD and its correlates in large samples, as well as the need to investigate biological markers (such as markers of inflammation) that could be associated with an increased risk of PTSD. From a preventive point of view, the usefulness of psychological first aid and the need to focus on high-risk populations, including healthcare workers, was emphasized. Finally, in terms of public outreach, the need to disseminate high quality information about trauma and its manifestations, and to work more closely with non-mental health services, were both discussed. These recommendations are undoubtedly of use to anyone involved in developing a robust response to PTSS/PTSD in the context of COVID-19. From a more immediately practical perspective, Dutheil et al. estimated that around 30–50% of the population affected by the COVID-19 pandemic might exhibit PTSS to some degree, and underlined the association between PTSD and suicide. They concluded that both these facets would need to be addressed through public health policy5. Finally, a paper comparing the impact of COVID-19 with the September 11, 2001 terrorist attack noted that the effects of COVID-19 in terms of psychological trauma were likely to be far more protracted, and that long-term follow-up care to address chronic PTSD would consequently be required23.\n\n\nConclusions\n\nThough the existing literature is confined to cross-sectional studies using screening tools, it is clear that significant PTSS affect a sizeable minority of individuals facing the consequences of the COVID-19 pandemic. Some of the variables associated with the severity of PTSS, such as female gender and separated/divorced marital status, have been reported in earlier studies of PTSD following disasters24,25; however, factors such as the presence of physical symptoms or poor perceived physical health are more likely to be specific to disease outbreaks. As there is an overlap between the factors identified in this review and those related to depressive or anxiety symptoms during the COVID-1926, they may represent general predisposing factors for psychological morbidity, rather than having a specific association with PTSD. Evidence from the only longitudinal study available to date suggests that PTSS are relatively stable over a period of four weeks. Literature from the SARS outbreak suggests that symptoms of PTSS persist in over half of those initially affected after over two years1; thus, further longitudinal studies are needed to examine the persistence of PTSS/PTSD in the aftermath of the COVID-19 pandemic and the factors associated with it.\n\nThis review is subject to certain important limitations. First, because of the limited literature in this field, this was a scoping review rather than a systematic review or meta-analysis, and its statistical rigour is accordingly limited. Second, all reviewed studies bar one were cross-sectional studies, allowing few conclusions to be drawn about the long-term course of PTSS in these individuals. Third, all studies relied on screening instruments, and hence were more likely to capture initial and acute responses to stress rather than PTSD itself, which may develop after several weeks or months. Fourth, most published studies were from a single geographical area, and their findings may not be entirely applicable to other countries and cultures.\n\nDespite these important caveats, there is enough evidence to suggest that PTSS is and will continue to be a significant problem as the COVID-19 pandemic continues to run its course. It is hoped that the findings summarized and reviewed above may be of use to researchers in this field, as well as to those involved in the identification, prevention, and management of post-traumatic stress related to COVID-19 at the clinic and community level.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "Acknowledgments\n\nThe author wishes to thank all the researchers and publishers who have made literature in this field easily accessible at a time of crisis.\n\n\nReferences\n\nMak IWC, Chu CM, Pan PC, et al.: Long-term psychiatric morbidities among SARS survivors. Gen Hosp Psychiatry. 2009; 31(4): 318–326. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee SM, Kang WS, Cho AR, et al.: Psychological impact of the 2015 MERS outbreak on hospital workers and quarantined hemodialysis patients. Compr Psychiatry. 2018; 87: 123–127. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaunder RG: Was SARS a mental health catastrophe? Gen Hosp Psychiatry. 2009; 31(4): 316–317. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJakovljevic M, Bjedov S, Jacksic N, et al.: COVID-19 pandemia and public and global mental health from the perspective of global health security. Psychiatr Danub. 2020; 32(1): 6–14. PubMed Abstract \| Publisher Full Text\n\nDutheil F, Mondillon L, Navel V: PTSD as the second tsunami of the SARS-CoV-2 pandemic. Psychol Med. 2020; 1–2. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoresh D, Brown AD: Traumatic stress in the age of COVID-19: a call to close critical gaps and adapt to new realities. Psychol Trauma. 2020; 12(4): 331–335. PubMed Abstract \| Publisher Full Text\n\nBrooks SK, Webster RK, Smith LE, et al.: The psychological impact of quarantine and how to reduce it: rapid review of the evidence. Lancet. 2020; 395(10227): 912–920. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChew QH, Wei KC, Vasoo S, et al.: Narrative synthesis of psychological and coping responses towards emerging infectious disease outbreaks in the general population: practical considerations for the COVID-19 pandemic. Singapore Med J. 2020. PubMed Abstract \| Publisher Full Text\n\nPfefferbaum B, North CS: Mental health and the COVID-19 pandemic. N Engl J Med. 2020; 1: 1–2. PubMed Abstract \| Publisher Full Text\n\nJenkins SR, Baird S: Secondary traumatic stress and vicarious trauma: a validational study. J Trauma Stress. 2002; 15(5): 423–32. PubMed Abstract \| Publisher Full Text\n\nWilliamson V, Murphy D, Greenberg N: COVID-19 and experiences of moral injury in front-line key workers. Occup Med (Lond). 2020; kqaa052. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGreen P: Risks to children and young people during COVID-19 pandemic. BMJ. 2020; 369: m1669. PubMed Abstract \| Publisher Full Text\n\nYang Y, Li W, Zhang Q, et al.: Mental health services for older adults in China during the COVID-19 outbreak. Lancet Psychiatry. 2020; 7(4): e19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYao H, Chen JH, Xu YF: Patients with mental health disorders in the COVID-19 epidemic. Lancet Psychiatry. 2020; 7(4): e21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTricco AC, Lillie E, Zarin W, et al.: PRISMA extension for scoping reviews (PRISMA-ScR): checklist and explanation. Ann Intern Med. 2018; 169(7): 467–473. PubMed Abstract \| Publisher Full Text\n\nTan W, Hao F, McIntyre RS, et al.: Is returning to work during the COVID-19 pandemic stressful? A study on immediate mental health status and psychoneuroimmunity prevention measures of Chinese workforce. Brain Behav Immun. 2020; S0889-1591(20)30603-6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChew NWS, Lee GKH, Tan BYQ, et al.: A multinational, multicentre study on the psychological outcomes and associated physical symptoms amongst healthcare workers during COVID-19 outbreak. Brain Behav Immun. 2020; S0889-1591(20)30523-7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHao F, Tan W, Jiang L, et al.: Do psychiatric patients experience more psychiatric symptoms during COVID-19 pandemic and lockdown? A case-control study with service and research implications for immunopsychiatry. Brain Behav Immun. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiang L, Ren H, Cao R, et al.: The effect of COVID-19 on youth mental health. Psychiatr Q. 2020; 1–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu N, Zhang F, Wei C, et al.: Prevalence and predictors of PTSS during COVID-19 in China hardest-hit areas: gender differences matter. Psychiatry Res. 2020; 288: 112921. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang C, Pan R, Wan X, et al.: A longitudinal study on the mental health of general population during the COVID-19 epidemic in China. Brain Behav Immun. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi Z, Ge J, Yang M, et al.: Vicarious traumatization in the general public, members, and non-members of medical teams aiding in COVID-19 control. Brain Behav Immun. 2020. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDePierro J, Lowe S, Katz C: Lessons learned from 9/11: mental health perspectives on the COVID-19 pandemic. Psychiatry Res. 2020; 288: 113024. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKun P, Tong X, Liu Y, et al.: What are the determinants of post-traumatic stress disorder: age, gender, ethnicity or other? Evidence from 2008 Wenchuan earthquake. Public Health. 2013; 127(7): 644–52. PubMed Abstract \| Publisher Full Text\n\nLowe SR, Bonumwezi JL, Valdespino-Hayden Z, et al.: Posttraumatic stress and depression in the aftermath of environmental disasters: a review of quantitative studies published in 2018. Curr Environ Health Rep. 2019; 6(4): 344–360. PubMed Abstract \| Publisher Full Text\n\nWang C, Pan R, Wan X, et al.: Immediate psychological responses and associated factors during the initial stage of the 2019 coronavirus disease (COVID-19) epidemic among the general population in China. Int J Environ Res Public Health. 2020; 17(5): 1729. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "77520", "date": "11 Mar 2021", "name": "Kang Sim", "expertise": [ "Reviewer Expertise Adult Psychiatry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 reads well, the methods are appropriate and the author should consider the following suggestions to improve the paper:\nThe rationale for choosing a scoping review over a systematic review can be briefly explained.\n\nIt would be good to state the exclusion criteria under the “Search Methodology” section as well.\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": "71936", "date": "01 Apr 2021", "name": "José María De la Roca-Chiapas", "expertise": [ "Reviewer Expertise Stress", "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\nPost-traumatic stress in the wake of the COVID-19 pandemic: a scoping review It is an interesting article that presents useful information on the subject of COVID-19. It is well presented, only it has a methodological error, which limits the results and its scope which I detail below:\nThe time of selection of the articles is very short, I would ask if it is your interest in indexing it, that the search will increase until September or October 2020, since a lot of information has been generated in this regard and not including it would be wasting the article.\n\nYou could also modify the title of the article and remove \"in the wake\".\n\nYou could also compare how it has occurred in different parts of the world since it is a global problem.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-675
https://f1000research.com/articles/9-449/v1	27 May 20	{ "type": "Research Article", "title": "Differential dynamics of early stages of platelet adhesion and spreading on collagen IV- and fibrinogen-coated surfaces", "authors": [ "Melanie B. Horev", "Yishaia Zabary", "Revital Zarka", "Simona Sorrentino", "Ohad Medalia", "Assaf Zaritsky", "Benjamin Geiger", "Melanie B. Horev", "Yishaia Zabary", "Revital Zarka", "Simona Sorrentino", "Ohad Medalia", "Assaf Zaritsky" ], "abstract": "Background: Upon wound formation, platelets adhere to the neighboring extracellular matrix and spread on it, a process which is critical for physiological wound healing. Multiple external factors, such as the molecular composition of the environment and its mechanical properties, play a key role in this process and direct its speed and outcome. Methods: We combined live cell imaging, quantitative interference reflection microscopy and cryo-electron tomography to characterize, at a single platelet level, the differential spatiotemporal dynamics of the adhesion process to fibrinogen- and collagen IV-functionalized surfaces. Results: Initially, platelets sense both substrates by transient rapid extensions of filopodia. On collagen IV, a short-term phase of filopodial extension is followed by lamellipodia-based spreading. This transition is preceded by the extension of a single or couple of microtubules into the platelet’s periphery and their apparent insertion into the core of the filopodia. On fibrinogen surfaces, the filopodia-to-lamellipodia transition was partial and microtubule extension was not observed leading to limited spreading, which could be restored by manganese or thrombin. Conclusions: Based on these results, we propose that interaction with collagen IV stimulate platelets to extend microtubules to peripheral filopodia, which in turn, enhances filopodial-to-lamellipodial transition and overall lamellipodia-based spreading. Fibrinogen, on the other hand, fails to induce these early microtubule extensions, leading to full lamellipodia spreading in only a fraction of the seeded platelets. We further suggest that activation of integrin αIIbβ3 is essential for filopodial-to-lamellipodial transition, based on the capacity of integrin activators to enhance lamellipodia spreading on fibrinogen.", "keywords": [ "Platelet spreading", "Microtubules", "Integrin αIIbβ3", "Type IV collagen", "Fibrinogen", "Interference Reflection Microscopy (IRM)", "Live cell imaging" ], "content": "Introduction\n\nMammalian platelets are small, anucleated megakaryocytes fragments that circulate in the bloodstream as single, smooth-surfaced discoid structures. Upon vascular injury, platelets are recruited to the wound, where they initiate the formation on the primary hemostatic plug, catalyze fibrin formation and facilitate wound healing. Their adhesion and subsequent spreading at the exposed extracellular matrix (ECM) play a key role in the healing process, which is modulated by a variety of external environmental factors such as additional plasma components, other ECM constituents, platelet-derived secreted factors and their specific membrane receptors1–4. In addition to the molecular composition of the environment, its physical properties, such as rigidity, topography and shear forces generated by the blood flow, also influence the platelets adhesion and spreading process.\n\nThese molecular interactions of the platelets with the microenvironment are governed by specific adhesion receptors, such as αIIbβ3 integrin, which bind to fibrinogen and participated in the formation of the fibrin clot, and α2β1 and GP-VI, that mediate platelet adhesion to collagen IV5–9. The molecular and physical cross-talk between platelets and their microenvironment was extensively investigated10–14, yet, given the complexity of the signaling pathways involved and their rapid activation, a comprehensive understanding of the platelet adhesion and activation process is still limited.\n\nPrevious studies have shown that platelets that are stimulated by a variety of agonists undergo radical morphological changes. The discoid shape, which is largely maintained by the circumferential marginal band of microtubules15–17, is affected by the agonists activation, and the cells acquire a spherical shape, extend filopodia and sheet-like lamellipodia18–20. The general process of platelet spreading on different surfaces was documented, yet how the platelets differentially respond to specific adhesive ligands is still unclear.\n\nIn this study, we compared the adhesion and spreading dynamics of isolated platelets on two distinct and physiologically-relevant ligands, namely collagen IV and fibrinogen, along a broad temporal scale, ranging from seconds to hours. We found that platelets in platelets-rich plasma (PRP-PL), and washed platelets (PLT) extend filopodia within a few seconds after reaching the close vicinity of fibrinogen or collagen IV surfaces, while still translocating along the surfaces. Stable adhesion to collagen IV or fibrinogen occurred roughly within a minute after filopodia extension, yet the mode of “immobilization” is clearly distinct; on fibrinogen the duration of “filopodial spreading” was shorter than on collagen IV (less than a minute, compared to ~2–3 minutes) and the onset of lamellipodia formation was apparent earlier (~1 minute, compared to over 2–3 minutes on collagen IV). It was nevertheless noted that only a fraction of the fibrinogen-adherent PRP-PLs proceeded to the lamellipodial spreading stage.\n\nUnexpectedly, the delayed lamellipodial spreading on collagen IV (but not on fibrinogen) was commonly preceded by an extension of a single or a couple of microtubules from the circumferential marginal band to the platelet periphery, that penetrated into the core of peripheral filopodia. These results suggest that microtubule extension to the periphery of collagen IV-adherent platelets might trigger filopodia-to-lamellipodia transition, and promote platelet spreading.\n\n\nMethods\n\nMaTek dishes were incubated with either 25 μg/ml human collagen IV (Sigma-Aldrich, Israel) in sterile PBS or with 50 μg/ml human fibrinogen (Sigma-Aldrich, Israel) in sterile PBS, overnight at 4C0. The human fibrinogen was rinsed twice in sterile PBS. Finally, blocking with BSA solution in sterile PBS (1mg/ml, 15 min incubation) was conducted, to reduce non- specific platelet attachment, followed by washing with PBS and with Tyrode’s buffer. The collagen IV was washed with DMEM- no additives (Sigma-Aldrich, Israel) for 3 × 30 min. To reduce acidity, it was then washed two times with Tyrode’s buffer. Both collagen IV and fibrinogen MaTek plates were subsided in Tyrode's-HEPES buffer pH 7.4 containing 5 mM dextrose/glucose.\n\nHuman platelets in platelet rich plasma (PRP-PL) were produced from freshly drawn blood of different healthy donors specifically for this study. As no Helsinki approval is required for such experiments, we have worked under the Weizmann Institute of Science Institutional Review Board (IRB) informed consent form, signed by each of the volunteers. The IRB application (375-1) was approved by the committee members.\n\nThe blood was collected into PT vacutainer tubes (containing sodium citrate). After 10 min of incubation at RT, the blood was centrifuged at 800 rpm for 10 min, and the supernatant, containing platelets and plasma was collected. Human washed platelets (PLT) were produced from freshly drawn blood of different healthy donors. The blood was collected into PT vacutainer tubes (containing sodium citrate). After 10 min of incubation at RT, the blood was centrifuged at 800 rpm for 10 min, and the supernatant, containing platelets and plasma was collected. Then an equal volume of Tyrode's solution pH 6.5 containing 5 mM dextrose, 0.2 µg/mL PGE1 and 1.0 U/mL apyrase was added and incubated for 5 min at RT, then the diluted PRP was centrifuged at 1500 rpm for 10 min to precipitate platelets. The supernatant was discarded and the pellet was re-suspended in Tyrode's solution, same volume as the discarded, at pH 6.5 containing 5 mM dextrose, 0.2 µg/mL PGE1 and 0.5 U/mL apyrase. The solution was incubated for 5 min at RT. Then it was centrifuged at 1500 rpm for 10 min. The supernatant was discarded and the pellet re-suspended in Tyrode's-HEPES buffer pH 7.4 containing 5 mM dextrose the same volume as the original PRP volume. The platelets were kept for 30–60 min at RT prior to use.\n\nFresh PRP-PL or PLT were seeded (10–15×106) on collagen IV and fibrinogen MaTek plates directly in the microscope environmental chamber at 37°C in humidified atmosphere of 5% CO2 and 95% air.\n\nPRP-PL and PLT attachment and spreading process on the surfaces was monitored in real time using the DeltaVision Elite® system, running softWoRx 6.0. As the system is equipped with an environmental box, PRPs in Tyrode’s solution were seeded directly onto the fibrinogen and collagen IV surfaces in the microscope environmental chamber (temperature- and CO2-controlled). Interference reflection contrast (IRM) time-lapse imaging of several fields per specimen was carried out using 100x/1.3 oil objective, at 5 seconds between frames. IRM, phase-contrast microscopy, and differential interference contrast (DIC) microscopy still imaging were used with a 100X objective (NA 1.3 and 1.4), a 60X (NA 1.42) and a 20X (NA 0.85) objective.\n\nSingle platelets were manually selected in the field-of-view. Since we were interested in minimizing the effects of neighboring platelets, we selected platelets that spread the earliest in their vicinity. For each platelet, we manually annotated a bounding box that captured the full spread platelet and recorded time points for the characterized stages in platelet spreading (defined in Results): onset, or initial appearance (time = 0); onset of filopodia; and lamellipodia spreading.\n\nTo visualize and quantify the change in the local focal plane during platelet spreading, we developed custom software using Matlab and Python (Software availability21). We defined the focal activity map as the time-derivative of a platelet’s IRM intensity. This was calculated for each time point as ΔIRMt = IRMt – IRMt+1. Local instantaneous attachment was encoded with positive values (time point t+1 is darker – closer to the substrate), while local instantaneous detachment corresponded to negative values (time point t+1 is brighter – further from the substrate). The focal activity map provides intuitive visual information on the platelet’s spatiotemporal focal-dynamics.\n\nThe integrated tapping activity of a platelet in a given frame in the image sequence was defined as the accumulation of all local alterations in focal plane in both attachment of detachment events. This measure was calculated as the average value of the absolute ΔIRM image pixels: Σi \|IRMt\|i, where i spans the platelet’s bounding-box’s pixels, and \|IRMt\|i is the corresponding pixel’s intensity (see Figure 2B). Importantly, this measure is relative; for example, more background pixels in a bounding box will dampen the magnitude regardless of the actual platelet activity.\n\n(A) DIC, time-lapse microscopy of platelets attaching to collagen IV at different time points. Black arrow denotes the platelet in question. Scale bar: 10 μm. (B) Phase-contrast microscopy of PRP-PL on fibrinogen. Left: Discoid shape; Right: Platelet with filopodial extension. Scale bar: 5 μm. (C) Phase-contrast microscopy of PLT on collagen IV. Left: Discoid shape; Right: Platelet with filopodial extension. Scale bar: 5 μm. (D) Platelet attachment to collagen IV or fibrinogen in percentage (%) over time. Time in seconds, on a logarithmic scale. Platelets on collagen IV attach faster that on fibrinogen. Wilcoxon rank-sum test, p-value < 0.015. N platelets: FBG=121, collagen IV=274. Error bars show Standard Error.\n\n(A) Platelet spreading viewed by IRM, and the corresponding focal activity map, ΔIRMt = IRMt – IRMt+1. Positive values (yellow) imply local attachment; negative values (blue) imply local detachment (bottom right). One filopodia initially attaching and detaching (black arrow). Scale bar 2 μm (B) Integrated tapping activity of platelets: the mean absolute value \|ΔIRM\| at every time point. X-axis: Time in seconds. Y-axis: Platelet mean activity. Red dotted lines separate the phases: background, prior to platelet attachment, filopodial spreading phase, lamellipodial spreading phase, and the fully spread phase. (C) Total number of pixels interacting with the surface over time. Time in seconds. (D) Accumulated attachment and detachment over time shown by activity map, yellow means more attachment events, blue means fewer attachment event. Images on the right correspond IRM images. Scale bar 2 um.\n\nTo follow the spreading process, we quantified the pixels that were touching or very close to the surface. We pooled all IRM pixel intensities across a platelet time-lapse sequence and used the Otsu algorithm22 to define a threshold that labels each pixel at every time point into ‘foreground’ or ‘background’. ‘Foreground’ pixels had low IRM intensities and were thus touching or very close to the surface, we termed these pixels as “interacting with the surface”. ‘Background’ pixels had higher IRM intensities and marked local regions that did not touch the substrate. We measured the number of pixels interacting with the surface over time during the spreading process (see Figure 2C). For each pixel, at each time frame, we accumulated the number of transitions from interaction to not-interacting with the surface from time 0 and until that time point (see Figure 2D).\n\nTo examine the spatiotemporal statistics of a platelet spreading, we identified a subgroup of active pixels. First, we manually marked a background region, an empty region close to the platelet, collected and pooled ΔIRM pixel intensities in the background throughout the platelet spreading process. Second, we used the accumulated background pixel intensity statistics to define thresholds of one standard deviation above or below the mean background pixel ΔIRM intensity. The subgroup of pixels that exceeded these ΔIRM thresholds were the more active pixels in terms of attaching/detaching activity. We calculated the fraction of active pixels that attached or detached for each time frame (see Figure 3A–C) and then overlaid the active pixels color-coded as attaching or detaching back to the image plane (see Figure 3D and E).\n\n(A–C) Temporal profile of the fraction of attachment (red) versus detachment (blue) events during platelet spreading: (A) collagen-IV substrate, (B) fibrinogen substrate, (C) control – region with no platelet. (D and E) Spatiotemporal dynamics of attachment (red) versus detachment (blue) events during platelet spreading. Time corresponds to the vertical dashed line in panels A–B correspondingly: (D) Collagen-IV substrate. Platelet spreading is characterized by attachment, initially throughout the cell and later at the platelet periphery. (E) Fibrinogen substrate. Platelet spreading is characterized by the attachment and detachment of entire filopodia structures (arrow heads).\n\nMn2+ (250 μM), molecule sn528 (αIIbβ3 integrin inhibitor; 10 μM) or thrombin (0.12 mg/ml) were added directly to the dishes containing fibrinogen or collagen IV in Tyrode's-HEPES buffer pH 7.4 containing 5 mM dextrose simultaneously as the fresh PRP-PL or PLT, which were seeded (10–15×106) in the microscope at 37°C in humidified atmosphere of 5% CO2 and 95% air.\n\nThe platelets were seeded for 10–20 min on fibrinogen or collagen IV. Subsequently, they were fixed by Karnovsky fixative (2% paraformaldehyde, 2.5 % glutaraldehyde). The solution was shaken well and incubated for 30 min. Then glycerol was added, spun down gently, and washed with PBS. The platelets were then suspended in PBS and glycerol (1:9), and the solution closed between coverslip and slide and imaged by phase contrast microscopy.\n\nNocodazole (10, 15 or 20 μM) was added to the platelets in solution immediately before seeding the platelets in the dish containing collagen IV in Tyrode's-HEPES buffer, pH 7.4, containing 5 mM dextrose. Platelets were seeded (10–15×106) in the microscope at 37°C in a humidified atmosphere of 5% CO2 and 95% air.\n\nVon Willebrand factor (2400 IU) Heamate P 1000 (CSL Behring, GmbH, Germany) was used in different concentrations (1:2, 1:5, 1:10), all yielding the same results. VWF was added directly to the dish containing collagen IV in Tyrode's-HEPES buffer, pH 7.4, containing 5 mM dextrose, simultaneously with the fresh PLT, which were seeded (10–15×106) in the microscope at 37°C in a humidified atmosphere of 5% CO2 and 95% air.\n\nPlatelets were seeded on collagen IV functionalized gold/silicon grids (R 1/4, 200 mesh, Quantifoli, Jena Germany). The grids were then vitrified by plunge freezing in liquid ethane and then immediately stored in liquid nitrogen until imaging. Data acquisition was performed using a FEI Titan Krios equipped with a quantum energy filter and a K2 Summit direct electron detector (Gatan, Pleasanton, USA). Tomograms were acquired with a magnification of 42,000× corresponding to a pixel size of 0.34 nm. The cumulative electron dose was ~70 electrons per ångström2. Tomograms were reconstructed with the TOM toolbox software package23.\n\nStatistical calculations were performed using Excel (version 14.6.8, 2011) and a STATISTICA® (version 13.1) software package, using the non-parametric Wilcoxon rank-sum test.\n\nImage analysis was performed using MATLAB (version 9.3), Amira/Avizo (version 3.1), motion correction software, and Fiji (ImageJ).\n\n\nResults\n\nWe chose to study two preparations of platelets, isolated from human donor blood: washed platelets (termed here PLT) and platelets in platelet-rich-plasma (termed PRP-PL). To create and test physiologically relevant environments, we plated platelets on tissue culture surfaces coated with either fibrinogen, an integral component of the blood clot; or collagen IV, a basement membrane molecule that platelets adhere to upon vessel injury. It is noted that this adhesive set-up is not entirely 'clean', as platelets secrete multiple biologically active molecules (e.g. fibrinogen). Furthermore plasma components in the PRP-PL may affect the experiments (for further discussion, see below). In this study, we have employed real time microscopy (primarily interference reflection optics (IRM) or differential interference contrast (DIC) for monitoring platelet-substrate interaction.\n\nPre-immobilization: Early events of the interaction of platelets with collagen IV-coated surfaces were visualized using live DIC imaging. With the microscope focused on the adhesive surface, we observed platelets that were located at the vicinity of the substrate, whose translocation was substantially attenuated relative to those still floating freely in the medium. Such loose interactions with the surface were noted within 15–25 seconds after reaching the substrate focal level (Figure 1A). We detected an active extension of “ventral filopodia” that was apparent as soon as 10 seconds after the platelet reached the surface (Figure 1A, black arrow inset) and persisted until the platelet became immotile. Upon turning immotile, the platelet continued to spread by extending stable substrate-attached filopodia, followed by lamellipodia, until reaching full spreading after 20–30 minutes (Figure 1A). We confirmed that some platelets extended filopodial extensions while being motile on collagen IV- or on fibrinogen-coated surfaces with chemical fixation and phase contrast microscopy imaging (Figure 1B–C, arrows). These results confirmed the existence of a filopodial extension prior to platelets immobilization and attachment to the substrate.\n\nPost immobilization, filopodial spreading: Platelets on collagen IV became immotile and attached to the surface at a faster pace than platelets on fibrinogen (Figure 1D). These results suggest that upon the onset of platelet immobilization and spreading, platelets can extend filopodial protrusions on both collagen IV and fibrinogen surfaces, but that platelet spreading is more effective on collagen IV surfaces.\n\nPost immobilization, lamellipodial spreading: The filopodial spreading, which was initiated still in the “mobile phase”, was followed by massive lamellipodial spreading (“filopodial-lamellipodial transition”), which was noted in the PRP-PL/fibrinogen system in ~55% of the platelets at about 1 minute after the first interaction with the surface. The onset of the filopodial-lamellipodial transition in the PLT/collagen IV system was delayed (~2–3 minutes) yet was apparent in a larger proportion of the platelet population ~75% (see results below).\n\nTo further characterize the spatiotemporal dynamics involved in platelet adhesion to collagen IV-coated surfaces, we used live cell IRM, where the light intensity is negatively correlated with the local platelet-surface proximity (Figure 2A, top). To quantitatively characterize the spatiotemporal dynamics at the single platelet level, we manually cropped single images of platelets and analyzed them by measuring the pixel-wise difference in the IRM intensities between consecutive frames of the movie, imaged at temporal resolution of 5 seconds, ΔIRMt = IRMt – IRMt+1, which we termed the focal activity map. This analysis provided us with quantitative information and visualization regarding the local platelet dynamics: positive values (yellow) imply local attachment, and negative values (blue) imply local detachment, in relation to the previous time frame (Figure 2A bottom). The brighter object observed at time = 0 was a characteristic feature of the IRM imaging for an unattached, but close to the surface object, and was consistently associated with a platelet appearing slightly above the IRM focal plane (Figure 2A top, arrowhead). This out-of-focus feature provides the first visual cue for a near-future platelet attaching and spreading. Small attachment regions could be observed upon platelet appearance, at ΔIRM0 (Figure 2A bottom, t = 0 sec., arrow). These local attachment regions corresponded to the future appearance of the first filopodia touching the surface (Figure 2A, t = 45 seconds, arrowhead) and appeared in 18 of 20 platelets that we analyzed. This observation suggests that platelets sense the substrate from a μm-scale distance by extending thin protrusions toward the substrate that will later develop into stable filopodia. The appearance of full filopodia was characterized by local instability; some regions in the platelet were attaching to the substrate, while others were detaching concurrently, we termed this initial stage in platelet spreading, as the filopodial spreading stage (Figure 2A, t = 45, 55 seconds). The filopodial stage was followed by a lamellipodial spreading stage, where local spatial instability was specifically observed at the platelet periphery, lamellipodia formed and platelet spreading preceded until reaching a fully spread morphology (Figure 2A, t = 390 seconds).\n\nTo examine the platelet spreading process in a more quantitative manner, we developed a custom pipeline to analyze the local attachment/detachment dynamics in IRM time-lapse and applied it to platelets spreading on collagen IV surfaces. Briefly, we overlaid a 3×3 pixel grid of bins on the ΔIRM image, and collected temporal statistics on the attachment/detachment activity within these bins (see Methods). The overall attachment/detachment activity of a platelet, which we call the platelet’s integrated tapping activity, was defined as the mean absolute value \|ΔIRM\| at every time point (Figure 2B). This measure constituted the integration of all local alterations in the focal plane, encompassing both attachment and detachment events. A platelet’s integrated tapping activity gradually increased during the filopodial stage, reaching a peak in activity, once this stage was complete. Platelet activity, then gradually decreased until it reached a plateau at the fully spread stage, which was above the initial integrated tapping activity before the platelet appeared. This was found to be consistent for most of the platelets measured (Extended data: Figure S124).\n\nWe next used a classical threshold-based segmentation method to separate the pixels of each time point into foreground and background pixels based on their IRM intensities22 (see Methods). “Foreground” pixels had lower IRM intensities and were thus touching or very close to the surface, we termed these pixels as “interacting with the surface”. The number of pixels interacting with the surface over time showed a slow increase during the filopodial stage, and increased rapidly during the lamellipodial stage until reaching the fully spread morphology (Figure 2C), this was consistent across several platelets (Extended data: Figure S224). By following the accumulated number of transitions from interaction to not-interacting with the surface at every pixel over time (see Methods), we observed that transitions occurred at the cell periphery as marked by no change in transitions accumulation in the platelet’s interior over time (Figure 2D). The “vein”-like trails from the platelet center to its periphery suggest that the same filopodium is responsible for the repetitive interaction with the surface prior to the more stable lamellipodia (Figure 2D; Extended data: Movie S124). Altogether, these data suggested that on collagen IV platelets extend filopodia that interact with the substrate by local attachment-detachment tapping events. This filopodia sensing coincides with a steady increase in lamellipodial area, which eventually leads to overall platelet spreading on collagen IV. We observed similar results for platelets spreading on fibrinogen (Extended data: Figure S324).\n\nOn one hand, filopodia appeared during platelet immobilization on both collagen IV and fibrinogen surfaces. On the other hand, platelet spreading is less effective on fibrinogen surfaces that were characterized by impaired transition to lamellipodial spreading. To pinpoint how local spatiotemporal dynamics lead to global differential platelet spreading we identified the subgroup of pixels that undergo significant attachment/detachment activity (see Methods). By plotting the fraction of the active pixels that undergo significant attachment (red) versus significant detachment (blue) we observed that platelet spreading on collagen IV was initially characterized by rapid and dominating attachment (Figure 3A). This is in contrast to the unstructured pattern that represents those platelets on fibrinogen that did not reach the lamellipodia-spreading phase (Figure 3B) or control regions with no platelets (Figure 3C). Overlaying these highly active regions on top of the IRM image revealed a striking differential pattern between platelet dynamics on collagen IV versus fibrinogen. While, platelets on collagen were characterized by attachment activity, initially throughout the cell body and later at the platelet periphery (Figure 3D; Extended data: Movie S224), platelets on fibrinogen were characterized by the attachment and detachment of entire filopodia structures (Figure 3E; Extended data: Movie S324). These results suggest that filopodia-to-lamellipodia transition is key for the effective spreading of platelets on collagen IV and less-efficient spreading on fibrinogen.\n\nA key step in platelet-substrate interaction is the transition from filopodial-to-lamellipodial spreading, which displayed different dynamics on the two surfaces (delayed by 1–2 minutes on collagen IV). In an attempt to understand the mechanism underlying the timing of this transition, we explored the cytoskeletal state of the platelets, particularly their microtubule (MT) dynamics by tagging live platelets with SIR-tubulin, which allows real-time monitoring of MT. These experiments clearly indicated that the vast majority (~94%, n=199) of platelets adhering to collagen IV commonly extend MTs towards the platelet periphery, where it interacts with and penetrates to a single, existing filopodium (Figure 4A and B, time point 175 seconds). The extension was not confined to a primary filopodia, suggesting that it is not involved in filopodia formation per se. Further analysis of the platelet spreading showed that the early-protruding lamellipodium usually extended from the location of the main MT-targeted filopodium (Figure 4C and E).\n\n(A) Platelet spreading every 5 seconds, as viewed by fluorescence microscopy (FM). Platelets tagged with tubulin are pictured in the upper row (B) In the row below, platelets are imaged by IRM scale bar 5 μm. (C) Platelet spreading on collagen IV at time point 170 sec. IRM (pink) overlay with SIR-tubulin (yellow). (D) Same as (C), at time point 270 sec. Lamellipodia formed where the SIR-tubulin extension is seen in filopodia (white arrows). (E) Scatter plot showing each experiment, how many platelet filopodia with microtubules extensions exhibit lamellipodia formation. X-axis number of MT containing filopodia forming lamellipodia, y-axis number of MT containing filopodia not forming lamellipodia. N experiments=4 and n platelets=58. (F) Washed platelets (PLT), on collagen IV and fibrinogen surfaces from left to right seen by SIR-tubulin and corresponding IRM, Sir-tubulin and corresponding IRM. Orange arrows indicate the dominant microtubule extension. Scale bar 10 μm. (G) IRM images from time-lapse movies of platelets treated with/without nocodazole. Scale bar: 10 μm. Time point: 60 min. Far right: boxplot showing platelet specific shape – filopodia or lamellipodia spread, with and without nocodazole treatment. We saw that there was a difference between platelets treated with nocodazole compared to the control in terms of the number of filopodia spread platelets and lamellipodia spread platelets. Wilcoxon sum rank test was performed * p-value < 0.01, N experiments=10, n platelets = 4669.\n\nIn platelets spreading on fibrinogen we did not observe microtubule extension into filopodia, suggesting that this phenomenon is collagen IV-specific. This observation, combined with the common failure of platelets adhering to fibrinogen, to undergo complete filopodial-to-lamellipodial switch, suggests that local microtubule extension to the platelet’s periphery plays a key role in the transition to lamellipodial spreading (Figure 4F). This notion is further supported by experiments showing that treatment of PLT/collagen IV with nocodazole impaired lamellipodia formation (Figure 4G), in agreement with25.\n\nThe MT-targeted filopodia from a random platelet revealed that microtubules entered the filopodia, but did not reach the tip of the filopodia as seen by cryo-electron tomography (Figure 5A and B). We observed an abundance of actin filaments (black arrows) in the filopodia, but only 1–2 microtubules that entered the filopodial core. Overall, we located MTs in ~58% of the filopodia we imaged (Figure 5C). From the 3D view we could appreciate the organized actin bundles at the tip of the filopodia, whilst more dispersed and organized in a network closer to the shaft (Figure 5D). We also observe a plentitude of trans-membrane, presumably including αIIbβ3 integrin, receptors (Figure 5E).\n\n(A) Low magnification image of a whole platelet with filopodial protrusions. Scale bar: 1 μm. (B) The same platelet as in (A), with a high magnification image of the filopodia in (A), black box. Slice from the 3D reconstructed volume. Visible is the membrane (orange arrow), microtubules (white arrow), and actin (black arrows). Scale bar: 140 nm. (C) Low magnification image of a representative platelet with filopodial extensions (black arrows). Scale bar: 1 μm (D) Rendered volume of the whole tomogram. Actin in yellow, microtubules in blue, membrane in grey, and surface receptors in light green. (E) Zoom-in view of (B), a slice from the 3D reconstructed volume. We are able to see the membrane (orange arrow), actin (black arrow), and surface receptors resembling integrins (blue arrow).\n\nWhen we first examined the interactions of both PLT and PRP-PL, with each of the two surfaces, striking differences in adhesion dynamics was found (Figure 6A and B). In the experiments presented in the previous sections of this article we have primarily compared the adhesion and spreading dynamics of PLT on collagen IV to those of PRP-PL to fibrinogen. The primary reason for this pairing of platelet type (PLT and PRP-PL) and matrix type (collagen IV and fibrinogen), respectively, was that switching these platelets-matrix pairs resulted in poor-to-no adhesion and spreading. Specifically, PLT on collagen IV were characterized by little aggregation, adhesion followed by filopodial, and then lamellipodial spreading as described above (see also Figure 6A). PRP-PL, on the other hand, failed to attach and spread on collagen (Figure 6B).\n\n(A–E) IRM imaging of platelet spreading for 1 hour. Scale bar: 10 μm. (A) PLT spreading on collagen IV surfaces. (B) PRP-PL spreading on collagen IV. (C) PRP-PL spreading on fibrinogen. (D) PLT spreading on fibrinogen. (E) PRP-PL and PLT spreading on fibrinogen and collagen IV, with and without the addition of plasma to the PLT, 1 hour post-platelet seeding. (F) Percentage of platelets in each morphological state, 1 hour post-platelet seeding.\n\nThis behavior was essentially mirror-imaged by PRP-PL, which attached to fibrinogen surfaces and proceeded by extending filopodia. Some of these platelets further developed lamellipodia and progressed to the fully spread state, while others remained in the filopodia state (Figure 6C). The attachment of PLT to fibrinogen was rather poor; only few platelets adhered to the matrix, and even fewer spread on it (Figure 6D).\n\nThe failure of PRP-PL to adhere to collagen IV was attributed to von Willebrand factor (VWF), which was present in the plasma, and effectively block collagen IV binding26,27. It was further shown that the addition of VWF to PLT preparations blocked the attachment and spreading (Figure 6E and F).\n\nThe limited ability of PLT to adhere and spread on fibrinogen is not entirely clear and could be attributed to the removal of VWF when the plasma is removed. VWF binding to integrin αIIbβ3 could play a role in this process28.\n\nThe overall spreading rate of PLT on collagen IV, from initiation to maximal extension, was ~ 2.5-fold slower than that of PRP-PL on fibrinogen (10 minutes, compared to 25 minutes, Figure 7A and B). Platelets on collagen IV exhibited longer filopodia and lamellipodia spreading phases, than platelets on fibrinogen (Figure 7C). The filopodia and lamellipodia spreading phases on both surfaces were demonstrated by scanning electron microscopy (Figure 7D–I).\n\n(A) IRM (100X) of a platelet (PLT) spreading on collagen IV; (left to right) filopodia tapping and fluctuations (0’’, 50’’), filopodia spreading (50’’, 200’’), lamellipodial protrusion (200’’), to the final, spread shape (25’). Scale bar: 10 μm. (B) IRM (100X) of a platelet (PRP-PL) spreading on fibrinogen; (left to right) filopodia tapping and fluctuations (0’’, 35’’), filopodia spreading (35’’, 85’’), lamellipodial protrusion (85’’) to the final, spread shape (8’). Scale bar: 10 μm. (C) Dynamics of platelet spreading. Graph showing the length of each of the phases described in (A) and (B). Wilcoxon rank sum test was used. p-value < 0.01, N platelets collagen IV=40, N platelets fibrinogen =40. Error bars show Standard Error. (D) SEM image (1.00 K X) of PLT on collagen IV. Scale bar: 10 μm. (E) SEM image (1.00 K X) of PLT on collagen IV, showing a filopodia spread platelet. (F) SEM image (1.00 X K) of PLT on collagen IV, showing a fully spread platelet (G) SEM image (1.00 K X) of PRP-PL on fibrinogen. Scale bar: 10 μm. (H) SEM image (1.00 K X) of PRP-PL on fibrinogen, showing a filopodia spread platelet. (I) SEM image (1.00 X K) of PRP-PL on fibrinogen, showing a fully spread platelet. Scale bar: 10 μm.\n\nBeyond the distinct spreading dynamics of PLT and PRP-PL on collagen IV and fibrinogen, their “spreading endpoints” were different. Within 1h after plating, 76% of PLT on collagen completed the filopodia-lamellipodia transition, while only 57% PRP-PL on fibrinogen failed to go through the filopodial-lamellipodial switch (Figure 8A). To explore the possibility that this arrest is due to insufficient activation of the αIIbβ3-receptor in the PRP-PL, we allowed these platelets to spread on fibrinogen for 30 minutes and then added the platelet activation agonists; Mn2+ or thrombin for additional 30 minutes (Figure 8B). These experiments clearly indicated that integrin (αIIbβ3) activation drives lamellipodia formation to full spreading (Figure 8B and C). To test the differential role of integrin αIIbβ3 in the adhesion and spreading on fibrinogen and collagen IV, we seeded PLT and PRP-PL on collagen IV and fibrinogen in the presence or absence of the αIIbβ3 inhibitor sn52829. This inhibitor had limited effect on the attachment and spreading on collagen while the adhesion to fibrinogen was completely blocked in line with earlier studies30 (Figure 9A and B).\n\n(A) PRP-PL on fibrinogen, imaged by IRM (100X magnification). PLT on collagen IV, imaged by IRM (100X magnification). Scale bar: 10 μm. Right graph: % distribution of filopodia and fully spread platelets. Platelet adhesion as a % of filopodia spread platelets vs. fully spread platelets, between PLT on collagen IV and PRP-PL on fibrinogen. Scale bar: 10 μm. Wilcoxon rank-sum test was used, p-value < 0.005 and ** p-value < 0.01. N experiments=8. N platelets: FBG=532, collagen IV=542. Error bars show Standard Error. (B) Platelets on the surfaces at 30 min. and 60 min. in the presence of manganese (Mn2+) and thrombin. Scale bar: 10 μm. (C) Overview over how many spread platelets per area (1 mm2) of the platelet preparations: PLT and PRP-PL on fibrinogen and collagen IV, in the absence and presence of the two agonist: Left Mn2+ treatment, Right thrombin treatment. Wilcoxon rank-sum test was used, * p-value < 0.05. N experiments=10. Error bars show Standard Error.\n\n(A) Inhibition of integrin αIIbβ3 by sn528. Effect of integrin αIIbβ3 inhibition shown by platelets on collagen IV and fibrinogen surfaces, with and without addition of the inhibitor sn528 (x-axis). Number of platelets per 1 mm2: y-axis. Wilcoxon rank-sum test was used, * p-value < 0.05 and ** p-value < 0.001. N experiments = 4. Error bars show Standard Error. (B) PLT on collagen IV with and without the addition of the sn528 ligand. PRP-PL on fibrinogen with and without addition of the sn528 ligand. Scale bar: 10 μm.\n\n\nDiscussion\n\nOur spatiotemporal analysis of platelet spreading demonstrated that platelets sense their environment well before they firmly attach to the functionalized surfaces. During these early spreading stages, the platelets undergo structural changes, manifested by the extension of filopodia, through which intermittent interactions with the surface are established, attenuate the platelets’ mobility, but do not block it (Figure 10A). These early adhesions, where the platelet changes from a discoid to a spherical shape, and after a short time (<1 min) extends actin-rich filopodia protrusions, followed by lamellipodia spreading was described in the literature, but their dynamic properties, and transition into stable adhesions were not characterized before20,31,32. Intermittent adhesions formed during the mobile phase (Figure 10B), led to immobilization of the adhering platelets, and triggered the subsequent spreading process. The dynamics of platelet spreading during the first minute after reaching the vicinity of the matrix was similar for platelets spreading on collagen or fibrinogen surfaces (Figure 10B, left from the “1 minute” time point). On the other hand, later events in the platelets’ response appeared matrix-dependent. Filopodial spreading persisted for longer periods and the onset of lamellipodial spreading was delayed on collagen IV. An additional and significant difference is the microtubule extension event, which is apparent only in the cells spreading on collagen. Taken together, these results suggest that distinct environmental signaling cues, induced by collagen IV and fibrinogen, are responsible for the differential late (~ 1–30 minutes) responses of the platelets.\n\n(A) The platelet (in orange) is floating as discoid shaped with the microtubule coil intact (red), it then extends filopodia while floating, hereby sensing the surface (yellow; collagen IV) and then exhibits oscillatory movements of attachment and detachment. It then attaches and sends out firmly attached protruding filopodia, one of them containing a dominant microtubule extension in the location where we see the primary lamellipodia forming and then continues to spread. (B) Overview of platelet temporal functional interplay. X-axis shows time, y-axis shows a diverse range of parameters under temporal investigation. Red shades correspond to either a mobile or immobile platelet. Blue indicates microtubule polymerization and the shades of green are the different morphological shapes of the platelet phases: discoid, filopodia and lamellipodial. The gradient illustrates if the certain parameter has more or less over all activity.\n\nTo monitor the early transition of the intermittent adhesions into more stable ones, we have employed and combined real-time DIC and IRM microscopy and developed dedicated image analysis techniques. Our dynamic adhesion data analysis revealed a novel pattern of oscillations, during which the platelet first moves closer to the surface, then further away, then closer again, the so-called tapping events. The frequency of these fluctuations was in the order of ~5–10 seconds, and especially obvious at the filopodial tips. These observations show that early platelet attachment events are oscillatory in nature, and might be coordinated by the platelet itself, yet the mechanism underlying these adhesion oscillations is still unknown. These integrated tapping active events were restricted to the early filopodial spreading stage, leveled out over the course of platelet spreading, and decreased toward the end of the lamellipodial extension period.\n\nTaken together these dynamic adhesion data suggest a model whereby platelets sense the surface through filopodial protrusions while still hovering over the surface (in a time-scale of a few-to-tens of seconds after reaching the surface vicinity). During this the platelet senses the substrate by intermittently touching it or ‘tapping’ on it, before they undergo extensive spreading (Figure 10A). We hypothesize that this ‘tapping’ process is a rather general mechanism for pre-adhesion sensing, that can play key role in focal complex or focal adhesion formation, as well as in the local organization of cytoskeletal components such as actin and microtubules. In this manner, the local sensing by filopodial protrusions may have a global effect, controlling platelet transformation from an early dynamic phase to a more mature and stable one.\n\nOne of the intriguing observations, reported here, is the apparent involvement of microtubules in the regulation of platelets’ spreading on collagen IV. These findings suggest that microtubules play a role in the progression of lamellipodia extension, in line with Waterman-Storer’s observation that microtubules are involved in actin-based protrusions at the leading-edge of migrating fibroblasts33. The absence of lamellipodial protrusions in most platelets adhering to fibrinogen might be attributed to the lack of microtubule-mediated stimulation. Previous studies demonstrated that the interplay between actin and microtubules is bidirectional, and can be directed by specific signaling pathways34. The functional significance of the microtubule extension and its relevance to the regulation of platelet spreading was reinforced by the demonstration that microtubule disruption by nocodazole blocked lamellipodia formation, in spreading PLTs.\n\nWhen we examined PLT on collagen IV and PRP-PL on fibrinogen, we see that the results indicate striking differences in adhesion dynamics. The primary difference of the two systems is in the receptors mediating the adhesion. In platelets the attachment to collagen IV follow the 2-site 2-step model where the initial attachment and tethering is orchestrated by the GPVI receptor, followed by inside-out activation of integrin α2β135,36. The collagen receptor complex GPVI-FcR γ-chain mediates platelet activation byligand-mediated clustering of the receptor which triggers an increase in Src family kinases (SFKs) activity and downstream tyrosine phosphorylation of enzymes, adaptors, and cytoskeletal proteins that collectively propagate the signal and coordinate platelet activation in the sense of cytoskeletal remodeling including actin protrusions, degranulation and integrin activation37,38.\n\nPRP-PL attached to fibrinogen presumably via integrin αIIbβ3, but also via GP-VI, as recent studies have shown39,40. Integrin αIIbβ3 can be activated by physiological agonists, such as ADP, thrombin or Manganese. When integrin αIIbβ3 is activated it transforms from a low- to high-affinity state. In this manner it binds fibrinogen and other ligands. When a ligand is bound, integrins cluster and the outside-in signaling cascade is stimulated. This drives essential platelet processes, such as attachment, spreading and thrombus formation. The exact mechanism involved in inside-out and outside-in signaling of integrin αIIbβ3 is not fully understood41,42. What we do know is that when integrin αIIbβ3 mediates outside-in signaling, it induces the tyrosine phosphorylation of multiple proteins. The SFKs plays a major role in these phosphorylation events. SFKs phosphorylate a host of signaling and cytoskeletal-associated proteins in platelets. This includes: phospholipase Cγ2 (PLCγ2), focal adhesion kinase (FAK), and adhesion- and degranulation-promoting adaptor protein (ADAP, also known as SLAP-130), which leads to their recruitment and/or activation42. The partial platelet spreading on collagen IV and especially fibrinogen, were caused by receptor inactivation, which was overcome by treatment with Mn2+ and thrombin. Indicating the necessity of in particular the αIIbβ3 integrin in the full platelet activation response.\n\nIt was clear to us that the adhesion to fibrinogen is strictly dependent on αIIbβ3, which we showed could be blocked by the specific inhibitor of this integrin, sn52810. Addition of this inhibitor to PLT plated on collagen had only limited effect, suggesting that the adhesion in that system is mediated mainly by the generic collagen IV receptors in platelets GP-VI and α2β1. This is consistent with the findings of Thornber et al were they defined the necessity of αIIbβ3 in lamellipodia progression for platelets on collagen related peptide, fibrinogen and thrombin30.\n\nWe believe the washed platelets between the surfaces cause differences in adhesion and spreading of PLT, affecting the microtubule filopodial extension, which may play a role in promoting the spreading for platelets on collagen IV. The lack of a MT extension may hinder lamellipodia spreading for PLT on fibrinogen. This would indicate a surface-specific receptor response and downstream signaling either via GP-VI and α2β1 for collagen IV or αIIbβ3 for fibrinogen.\n\n\nConclusions\n\nThis study demonstrates the extraordinary capacity of human platelets to differentially sense and respond to distinct physiological microenvironments - collagen IV and fibrinogen. We show that the adhesion and spreading on these surfaces is a highly complex process that combines selective receptor-mediated interactions, and the triggering of downstream cytoskeletal reorganization processes, which reinforce the platelet’s interaction with the external surfaces via specific membrane protrusions – filipodia and lamellipodia. These spatially and temporally coordinated events take place in a time scale of seconds to minutes, and involve integrin αIIbβ3 activation, for platelets adhering to fibrinogen and microtubule-based activation of lamellipodia in platelets adhering to collagen IV.\n\n\nData availability\n\nZenodo: IRM raw data (video format) and dataset (csv) supporting platelet attachment to collagen IV or fibrinogen in percentage over time (related to Figure 1), https://doi.org/10.5281/zenodo.377481943.\n\nZenodo: Raw data, temporal profiling for platelet spreading dynamics (related to Figure 3). https://doi.org/10.5281/zenodo.377482344.\n\nZenodo: Raw data for microtubule extension IRM images (videos) and raw data set (csv) (related to Figure 4), https://doi.org/10.5281/zenodo.377482745.\n\nZenodo: Raw data (IRM videos) of Nocodazole experiments (videos) and raw dataset for statistical purposes (csv) (related to Figure 4), https://doi.org/10.5281/zenodo.377483546.\n\nZenodo: Nocodazole experiment low mag images, IRM, raw data. Platelets fixed, imaged by IRM in low magnification for counting purposes. Platelets are either control or treated with nocodazole, https://doi.org/10.5281/zenodo.377484347.\n\nZenodo: Raw data to support percentage of platelets in each morphological state, 1 hour post-platelet seeding (related to Figure 8), https://doi.org/10.5281/zenodo.377484548.\n\nZenodo: Dynamics of platelet spreading over time with/without treatments with manganese and thrombin (related to Figure 8). Raw images of platelets treated with and without Manganese and thrombin (tif, jpegs) and raw data set (csv), https://doi.org/10.5281/zenodo.377484949.\n\nZenodo: Un-cropped and unedited images/movies for all (DIC, movies, cryo-ET, SEM images). https://doi.org/10.5281/zenodo.377343750.\n\nFigshare: Differential dynamics of early stages of platelet adhesion and spreading on collagen IV- and fibrinogen-coated surfaces, https://doi.org/10.6084/m9.figshare.c.494473824.\n\nThis project contains the following extended data:\n\nFigure S1. Platelet integrated activity. Integrated activity of platelets: the mean absolute value \|ΔIRM\| at every time point. X-axis: Time in seconds. Y-axis: Platelet mean activity. Red dotted lines separate the phases: background, prior to platelet attachment, filopodial spreading phase, lamellipodial spreading phase, and the fully spread phase.\n\nFigure S2. Interactions with the surface for collagen IV and fibrinogen. The number of pixels interacting with the surface over time for the surfaces collagen IV and fibrinogen. Time in seconds.\n\nFigure S3. Quantification and image analysis of platelet spreading, based on IRM live imaging for fibrinogen. (A) Platelet spreading viewed by IRM, and the corresponding focal activity map, ΔIRMt = IRMt – IRMt+1. Positive values (yellow) imply local attachment; negative values (blue) imply local detachment (bottom right). One filopodia initially attaching and detaching (black arrow). Scale bar 2 μm (B) Integrated tapping activity of platelets: the mean absolute value \|ΔIRM\| at every time point. X-axis: Time in seconds. Y-axis: Platelet mean activity. Red dotted lines separate the phases: background, prior to platelet attachment, filopodial spreading phase, lamellipodial spreading phase, and the fully spread phase. (C) Total number of pixels interacting with the surface over time. Time in seconds. (D) Accumulated attachment and detachment over time shown by activity map, yellow means more attachment events, blue means fewer attachment event. Right images, correspond IRM images. Scale bar 2 um.\n\nMovie S1. Shows the accumulated number of transitions from interaction to not interacting with the surface at every pixel over time.\n\nMovie S2. Shows an overlay of the highly active regions on top of the IRM images over time on collagen IV.\n\nMovie S3. Shows an overlay of the highly active regions on top of the IRM images over time on fibrinogen.\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\nIRM spreading dynamics source code available from: https://github.com/assafZaritskyLab/IRM-Spreading-Dynamics\n\nArchived source code as at time of publication: https://doi.org/10.5281/zenodo.377050621\n\nLicense: GNU General Public License v3.0", "appendix": "References\n\nRuggeri ZM: Platelets in atherothrombosis. Nat Med. 2002; 8(11): 1227–1234. PubMed Abstract \| Publisher Full Text\n\nVarga-Szabo D, Pleines I, Nieswandt B: Cell adhesion mechanisms in platelets. Arterioscler Thromb Vasc Biol. 2008; 28(3): 403–412. PubMed Abstract \| Publisher Full Text\n\nCabodi S, Di Stefano P, Leal Mdel P, et al.: Integrins and signal transduction. Adv Exp Med Biol. 2010; 674: 43–54. 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Handb Exp Pharmacol. 2012; (210): 3–22. PubMed Abstract \| Publisher Full Text\n\nZarka R, Horev MB, Volberg T, et al.: Differential Modulation of Platelet Adhesion and Spreading by Adhesive Ligand Density. Nano Lett. 2019; 19(3): 1418–1427. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJirousková M, Jaiswal JK, Coller BS: Ligand density dramatically affects integrin alpha IIb beta 3-mediated platelet signaling and spreading. Blood. 2007; 109(12): 5260–5269. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVlodavsky I, Eldor A, HyAm E, et al.: Platelet interaction with the extracellular matrix produced by cultured endothelial cells: a model to study the thrombogenicity of isolated subendothelial basal lamina. Thromb Res. 1982; 28(2): 179–191. PubMed Abstract \| Publisher Full Text\n\nLickert S, Sorrentino S, Studt JD, et al.: Morphometric analysis of spread platelets identifies integrin αIIbβ3-specific contractile phenotype. Sci Rep. 2018; 8(1): 5428. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee D, Fong KP, King MR, et al.: Differential dynamics of platelet contact and spreading. Biophys J. 2012; 102(3): 472–482. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaydon GB, Taylor DA: Microtubules in hamster platelets. J Cell Biol. 1965; 26(2): 673–676. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBehnke O: Further studies on microtubules. A marginal bundle in human and rat thrombocytes. J Ultrastruct Res. 1965; 13(5): 469–477. PubMed Abstract \| Publisher Full Text\n\nItaliano JE Jr, Richardson JL, Patel-Hett S, et al.: Angiogenesis is regulated by a novel mechanism: pro- and antiangiogenic proteins are organized into separate platelet alpha granules and differentially released. Blood. 2008; 111(3): 1227–1233. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWhite JG: Fine structural alterations induced in platelets by adenosine diphosphate. Blood. 1968; 31(5): 604–622. PubMed Abstract \| Publisher Full Text\n\nHartwig JH: Mechanisms of actin rearrangements mediating platelet activation. J Cell Biol. 1992; 118(6): 1421–1442. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBearer EL, Prakash JM, Li Z: Actin dynamics in platelets. Int Rev Cytol. 2002; 217: 137–182. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nassafZaritskyLab: assafZaritskyLab/IRM-Spreading-Dynamics: IRM-Spreading-Dynamics-BGU-ReleaseV1.0 (Version v1.0.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3770506\n\nOtsu N: A Threshold Selection Method from Gray-Level Histograms. IEEE T Syst Man Cy. 1979; 9(1): 62–66. Publisher Full Text\n\nNickell S, Förster F, Linaroudis A, et al.: TOM software toolbox: acquisition and analysis for electron tomography. J Struct Biol. 2005; 149(3): 227–234. PubMed Abstract \| Publisher Full Text\n\nHorev M: Supplementary data. figshare. 2020. https://doi.org/10.6084/m9.figshare.c.4944738.v1\n\nVasquez RJ, Howell B, Yvon AM, et al.: Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro. Mol Biol Cell. 1997; 8(6): 973–985. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFlood VH, Schlauderaff AC, Haberichter SL, et al.: Crucial role for the VWF A1 domain in binding to type IV collagen. Blood. 2015; 125(14): 2297–2304. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFuchs B, Budde U, Schulz A, et al.: Flow-based measurements of von Willebrand factor (VWF) function: binding to collagen and platelet adhesion under physiological shear rate. Thromb Res. 2010; 125(3): 239–245. PubMed Abstract \| Publisher Full Text\n\nOzaki Y, Suzuki-Inoue K, Inoue O: Platelet receptors activated via mulitmerization: glycoprotein VI, GPIb-IX-V, and CLEC-2. J Thromb Haemost. 2013; 11(Suppl 1): 330–339. PubMed Abstract \| Publisher Full Text\n\nWeiss M, Frohnmayer JP, Benk LT, et al.: Sequential bottom-up assembly of mechanically stabilized synthetic cells by microfluidics. Nat Mater. 2018; 17(1): 89–96. PubMed Abstract \| Publisher Full Text\n\nThornber K, McCarty OJ, Watson SP, et al.: Distinct but critical roles for integrin alphaIIbbeta3 in platelet lamellipodia formation on fibrinogen, collagen-related peptide and thrombin. FEBS J. 2006; 273(22): 5032–5043. PubMed Abstract \| Publisher Full Text\n\nMattila PK, Lappalainen P: Filopodia: molecular architecture and cellular functions. Nat Rev Mol Cell Biol. 2008; 9(6): 446–454. PubMed Abstract \| Publisher Full Text\n\nSandmann R, Koster S: Topographic Cues Reveal Two Distinct Spreading Mechanisms in Blood Platelets. Sci Rep. 2016; 6: 22357. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWaterman-Storer CM, Worthylake RA, Liu BP, et al.: Microtubule growth activates Rac1 to promote lamellipodial protrusion in fibroblasts. Nat Cell Biol. 1999; 1(1): 45–50. PubMed Abstract \| Publisher Full Text\n\nEtienne-Manneville S: Actin and microtubules in cell motility: which one is in control? Traffic. 2004; 5(7): 470–477. PubMed Abstract \| Publisher Full Text\n\nNieswandt B, Brakebusch C, Bergmeier W, et al.: Glycoprotein VI but not alpha2beta1 integrin is essential for platelet interaction with collagen. EMBO J. 2001; 20(9): 2120–2130. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNieswandt B, Watson SP: Platelet-collagen interaction: is GPVI the central receptor? Blood. 2003; 102(2): 449–461. PubMed Abstract \| Publisher Full Text\n\nSenis YA, Mazharian A, Mori J: Src family kinases: at the forefront of platelet activation. Blood. 2014; 124(13): 2013–2024. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInoue O, Suzuki-Inoue K, Dean WL, et al.: Integrin alpha2beta1 mediates outside-in regulation of platelet spreading on collagen through activation of Src kinases and PLCgamma2. J Cell Biol. 2003; 160(5): 769–780. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMangin PH, Onselaer MB, Receveur N, et al.: Immobilized fibrinogen activates human platelets through glycoprotein VI. Haematologica. 2018; 103(5): 898–907. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSavage B, Ruggeri ZM: Selective recognition of adhesive sites in surface-bound fibrinogen by glycoprotein IIb-IIIa on nonactivated platelets. J Biol Chem. 1991; 266(17): 11227–11233. PubMed Abstract\n\nBennett JS, Berger BW, Billings PC: The structure and function of platelet integrins. J Thromb Haemost. 2009; 7(Suppl 1): 200–205. PubMed Abstract \| Publisher Full Text\n\nDurrant TN, van den Bosch MT, Hers I: Integrin αIIbβ3 outside-in signaling. Blood. 2017; 130(14): 1607–1619. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHorev M: PhD. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3774819\n\nHorev MB: PhD [Data set]. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3774823\n\nHorev MB: PhD. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3774827\n\nHorev MB: PhD. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3774835\n\nHorev MB: PhD. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3774843\n\nHorev MB: PhD. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3774845\n\nHorev MB: PhD. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3774849\n\nHorev MB: PhD. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3773437" }	[ { "id": "64004", "date": "01 Jun 2020", "name": "Sandra Citi", "expertise": [ "Reviewer Expertise Cell-cell junctions", "cytoskeleton." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 on the initial phases of adhesion and spreading of platelets on two different substrates, collagen IV and fibrinogen. The authors report that on collagen IV a short-term phase of filopodial extension is followed by lamellipodia based-spreading, correlating with the insertion of 1-2 microtubules at the core of the filopodium. On fibrinogen, in contrast, there was only partial filopodia-to-lamellipodia transition, and microtubule extension was not observed. The quantification and image analysis of platelet spreading based on interference reflection microscopy, allowing a temporal profiling of attachment vs detachment and cryo-EM data are particularly interesting. The methods are state-of-the art, the results convincing, and the conclusions are sound.\nThe results should be discussed in the context of previous work on the distribution of microtubules in resting platelets and platelet filopodia (Patel-Hett et al. Blood, 20081).\n\nSome of the Figures should be improved for clarity. Figure 1: label panels A-C on the sides. Figure 3 D-E: label panels on the side (D- collagen, E-fibrinogen). Figures 7, 8 and 9: please use dot plots and not bar-graphs. Figure 10 label Panels (A and B), and schematic panel (left) for fibrinogen, to make the difference clearer (only collagen is shown).\nIn the overview of platelet temporal functional interplay (Fig 10) it is not clear why the lamellipodia shape phase is there, since the filopodia-to-lamellipodia transition is partial?\nTypos: 4°C not 4C° in first paragraph of Methods, by ligand not byligand in 5th paragraph Discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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": "5663", "date": "03 Jul 2020", "name": "Benny Geiger", "role": "Author Response", "response": "For Sandra Citi Thank, Sandra, for the useful comments, suggestions, corrections and thoughts. 1) We have further addressed, in the discussion MTs organization and dynamics in platelets, with the relevant reference. 2) We have added labels in fig. 10 (A and B) and we have referred to the difference between collagen and fibrinogen in the figure legend more clearly. Since the figures would be very similar we thought it to be better to address the issue in this manner. 3) Typos were fixed" } ] }, { "id": "64005", "date": "08 Jun 2020", "name": "Kenneth Yamada", "expertise": [ "Reviewer Expertise Cell-extracellular matrix interactions", "cell migration", "branching morphogenesis" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 provides nicely quantitative descriptions of the early steps of platelet attachment to either collagen IV or fibrinogen. As such, it will provide valuable information for the field and stimulate additional research in this area, e.g., concerning the regulation of filopodial activity during spreading. There are several aspects of this excellent study that would be strengthened by the addition of either limited amounts of new data or at least some additional discussion.\nThis study focuses on platelet attachment to extracellular proteins absorbed to a substrate, presumably modeling the interaction of platelets with basement membrane or a blood clot. Although the interaction of platelets with fibrinogen has been studied extensively in solution after activation by molecules such as thrombin during the process of platelet aggregation, this study seems to focus on interactions with an immobilized substrate. In that regard, the authors might consider that fibrin rather than fibrinogen would be the more physiological substrate. The results of initial attachment and spreading might be different on fibrin, because it is known that platelets can interact via integrin receptors more effectively to fibrin compared to fibrinogen [e.g., see PMID 26867579 and also 290215781,2]. Although this reviewer does not feel it is essential, it would be quite interesting to see whether the key findings concerning platelet attachment to fibrinogen are different when the substrate is fibrin. Alternatively, the authors could discuss this point about the types of substrate in vivo.\n\nThe differences in response to collagen IV and fibrinogen depending on whether they were in PRP or in a salt solution were extremely dramatic. One question that should ideally be discussed involves the physiological interpretation of the blocking of adhesion by von Willebrand factor. Does this mean that attachment to exposed collagen IV in damaged basement membranes in vivo is unlikely? Some additional discussion of these surprising findings would be helpful.\n\nThe images of apparent surface receptors in Figure 5 are quite impressive. However, it is not clear whether the authors can call them “surface receptors” without some additional information. For example, were morphological differences observed consistent with the folded and open erect configurations of integrins before and after platelet activation?\n\nMinor comments:\nA. In Figure 2D, the figure legend refers to “detachment” over time, yet the heat map coding seems to refer to more or fewer attachment events, so how was detachment represented in this figure?\nB. In Figure 4, what were the substrates used for panel sequences A and B?\nC. In Figure 7, and the text, how was a “filopodia spread platelet” defined?\nD. Figure 9A, do the authors happen to have any information on which receptor was used by activated platelets to spread on collagen IV after the treatment with sn528?\nE. In the summary shown in Figure 10, although this is not at all essential, the authors might consider including information on the conditions used for their platelets, specifically PLT vs. PRP-PL; if not practical, this information should be indicated in the figure legend so that readers will not assume that the platelets were compared under identical conditions on the two different substrates.\nF. The dishes used in this study were presumably from MatTek rather than from MaTek.\nG. Very minor technical questions: Was the concentration of thrombin used actually 0.12 mg/ml? What was the purpose of the glycerol added to chemically fixed platelets?\n\nIs the work 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": "5664", "date": "03 Jul 2020", "name": "Benny Geiger", "role": "Author Response", "response": "For Kenneth YamadaThank you, Ken, for the insightful questions, suggestions and additions to discussion. 1) We did compare the platelet “2D adhesion” to substrate-attached fibrinogen- structures where to “3D adhesion” to fibrin fibers. In the former we can see both filopodial and lamellipodial spreading as described in great detail throughout the paper. Platelets spreading on fibrin fibers was predominantly aligned along the underlying fibers, with essentially no sign for lamellipodia. These differences can be seen here.2) We have addressed the point you made in the relevant section of the revised manuscript.We do not propose that attachment to exposed collagen IV in damaged basement membranes in vivo is unlikely. The difference between the in vivo setting, where flow exists, which reinforces matrix adhesion. In our work the adhesion occurs under static condition where no or very little attachment occurs. This is attributable to the fact that VWF-platelet binding is facilitated by the A1 domain of VWF, which binds to the platelet receptor GPIb/IX/V. This binding requires immobilization followed by shear force, in order to be exposed and available for binding. Since we work under static experimental conditions, this binding doesn’t take place.3) In previous studies, Ohad Medalia’s group conducted gold labeling experiments to localize integrins (Dahan et al. 2018, Tamir et al. 2016) and glycoprotein 1b receptors (Stivala S, et al. Haematologica 2020) within platelets. They showed that many of the densities observed by cryo-ET, in platelets protrusions, are indeed integrins. These references were introduced and added to the paper. Moreover, cryo-ET of mouse platelets lacking αIIbb3 (genetically removed) showed no densities around the plasma membrane (Sorrentino et al. in preparations).All the minor comments were addressed. Specifically: a) The glycerol was added to the fixation solution in order to provide a protective environment around the sensitive platelet filopodia. b) The ‘filopodia spread platelet’ is a platelet with filopodia that does not display lamellipodial extensions and stays in this shape for the entire experiment duration." } ] }, { "id": "64006", "date": "10 Jun 2020", "name": "Ulrich Schwarz", "expertise": [ "Reviewer Expertise theoretical biophysics", "cellular biophysics", "mechanobiology", "cell adhesion", "cytoskeleton" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 performed a quantitative analysis of platelet spreading on either collagen or fibrinogen coated surfaces. The battery of methods applied is pretty impressive: DIC, RICM (also called IRM), FM (with SIR-tubulin), cryoEM, SEM and quantitative image processing. Together these measurements provide interesting new insight into an important part of wound healing. In particular, this study reveals a surprisingly large role of microtubules, which are known to be important for platelet biology due to the marginal band, but here are also shown to insert into actin-based filopodia, which probe the surface in a “tapping” phase and later initiate lamellipodia. This study also shows the important role of different integrins selected by different matrix components (or blocked by plasma components like the VWF), leading to more complete spreading on collagen. All in all this study is an important advance for a quantitative understanding of the platelet spreading part of wound healing.\n\nThere are a few issues that should be addressed in a revision:\nPlatelet activation: the time point cero for the start of spreading seems to be defined by contact with the matrix-decorated surface. Later in the study additional activation by thrombin is mentioned. However, usually platelets are activated by thrombin or ADP at the beginning, and with variable doses. It should be explained why the study has been performed without such design (or if the reviewer misunderstood this part).\n\nRelation to marginal band (MB): the new data on the microtubules is intriguing, but the relation to the marginal band is not clear. It is mentioned that the microtubules inserted into the filopodia arise from the MB. Can this be explained in more detail? Is this process regulated, e.g. by changing motor activity in the MB? It is well known that the MB coils after activation (compare e.g. Diagouraga et al. J Cell Biol 204: 177–185, 20141) and I wonder if this can be seen in Fig. 4.\n\nRole of actomyosin: coiling of the MB is related to cortical contractility (compare above reference). Moreover it is well known that platelets also form stress fibers (compare Tanaka and Itoh, J Struct Biol 124: 13–41, 19982) and recently it has been shown with traction force microscopy that they build up actomyosin-based traction forces with a time delay in regard to area spreading (Hanke et al. Soft Matter 14:6571-6581, 20183). For the general understanding of how the different processes are orchestrated during platelet spreading (as summarized in Fig. 10), it would be helpful to also explain the role of actomyosin contractility.\n\nComparison collagen versus fibrinogen: for most of the study these two conditions are treated symmetrically, but important Fig. 2 only shows data for collagen. I suggest to show the same analysis also for fibrinogen, if possible. This way it would also fit better to Fig. 3.\n\nIs the work 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": "5665", "date": "03 Jul 2020", "name": "Benny Geiger", "role": "Author Response", "response": "For Ulrich Schwarz Thank you Uli for the insightful comments, questions raised and suggestions. 1) Good point, we decided upon this particular set-up because we wanted to observe the platelet behavior before the addition of an agonist (Mn++ or thrombin). 2) Thanks for relating to the marginal band of microtubules. The marginal band may be seen in figure 4 as suggested, and the relevant live-cell imaging is added here. We have found a good example, addressing the question. Please see movie accessible here. We do believe we can see the MB coiling here, with an extending microtubule. 3) We didn't include it in the study. Treatment with Blebbistatin affected the organization of actin and focal adhesion like structures, but the Blebbistatin-treated platelets effect on MT extension was not tested. 4) The data is available as fig. S3, we felt it would be to cluttered to have both in one main figure." } ] } ]	1	https://f1000research.com/articles/9-449
https://f1000research.com/articles/9-671/v1	02 Jul 20	{ "type": "Opinion Article", "title": "Rational use of SARS-CoV-2 polymerase chain reaction tests within institutions caring for the vulnerable", "authors": [ "Tom A. Yates", "Graham S. Cooke", "Peter MacPherson", "Graham S. Cooke", "Peter MacPherson" ], "abstract": "Institutions such as hospitals and nursing or long-stay residential homes accommodate individuals at considerable risk of mortality should they acquire SARS-CoV-2 infection. In these settings, polymerase chain reaction tests play a central role in infection prevention and control. Here, we argue that both false negative and false positive tests are possible and that careful consideration of the prior probability of infection and of test characteristics are needed to prevent harm. We outline evidence suggesting that regular systematic testing of asymptomatic and pre-symptomatic individuals could play an important role in reducing transmission of SARS-CoV-2 within institutions. We discuss how such a programme might be organised, arguing that frequent testing and rapid reporting of results are particularly important. We highlight studies demonstrating that polymerase chain reaction testing of pooled samples can be undertaken with acceptable loss of sensitivity, and advocate such an approach where test capacity is limited. We provide an approach to calculating the most efficient pool size. Given the current limitations of tests for SARS-CoV-2 infection, physical distancing and meticulous infection prevention and control will remain essential in institutions caring for vulnerable people.", "keywords": [ "COVID-19", "SARS Coronavirus 2", "Infection Prevention and Control", "Clinical Diagnostics", "Epidemiology" ], "content": "Introduction\n\nWhilst there are significant limitations to the prognostic models that have been published to date1, the association between older age and death in SARS-CoV-2 infection is both clear and striking – the infection fatality rate for people in their 80’s has been estimated at 7.8%2. Infection prevention and control (IPC) is therefore crucial within institutions that bring together many such individuals, such as hospitals and nursing or long-stay residential homes.\n\nHere we discuss the use of polymerase chain reaction (PCR) tests within institutions caring for vulnerable individuals. We emphasise that consideration of test characteristics and the prior probability of SARS-CoV-2 infection is essential in making safe decisions based on PCR results. For example, decisions regarding the allocation of side rooms, whether patients can be cohorted in COVID or non-COVID spaces, and whether staff should be at work. We discuss the different uses of PCR tests, and consider which testing strategies should be prioritised. We explore how pooling samples prior to testing can result in a more efficient use of test capacity, and provide an optimised approach to selecting the number of samples to pool. Finally, we suggest how staff testing programmes for institutional care settings might be efficiently organised. We do not discuss disease surveillance or the role of testing for disease control in the wider community, but would point readers to recent modelling studies3,4. We only briefly discuss serological tests, as these are not yet in routine use. Our intended audience are those designing testing strategies in health and social care settings, as well as individuals in these settings acting on test results.\n\n\nEpidemiology and importance of transmission dynamics for institutional care settings\n\nHigh rates of SARS-CoV-2 infection with consequent morbidity and mortality have been observed in healthcare facilities and social care settings. Two infection prevalence surveys conducted in April and May 2020 in England found 1.33% and 1.73% of people working in ‘patient-facing healthcare or resident-facing social care roles’ had positive SARS-CoV-2 PCRs as compared with 0.22% and 0.38% of people not working in such roles5,6. Over much of this period, health and social care workers were one of the few groups permitted to leave their homes to work.\n\nA transmission model parameterised to a typical hospital in the United Kingdom suggests that up to 20% of infections in patients and 89% of infections in healthcare workers may be a result of nosocomial transmission7. Consistent with that, SARS-CoV-2 infections in healthcare workers and staff have been observed to cluster on particular hospital wards8 and a large proportion of hospital staff working in particularly exposed roles, such as housekeepers (34.5%) and those working in acute medicine (33.3%), show serological evidence of having been infected during the initial weeks of the pandemic9.\n\nLarge transmission clusters of SARS-CoV-2 infections have been seen in hospitals and elderly care facilities, along with worker dormitories and ships10. In one detailed case report incorporating phylogenetic analysis, a single introduction of SARS-CoV-2 into a private hospital in South Africa led to 80 staff members and 39 patients becoming infected, with 15 patient deaths11. The consequences of transmission within the social care sector have, perhaps, been even more severe. In one well-described outbreak in a ‘skilled nursing facility’, 57/89 (64%) of residents were infected, of whom fifteen died; a case fatality rate of 26%12. Viral sequence data were consistent with two separate introductions of SARS-CoV-2 into the facility12.\n\nA key feature of the epidemiology of SARS-CoV-2 transmission is that viral shedding begins 2–3 days prior to symptom onset13. A high proportion of transmission is thought to occur prior to symptom onset, estimated at 40–50% in studies from different settings13–16. Disease control strategies that rely on isolation once symptoms develop are, therefore, unlikely to be sufficient17.\n\nWhilst a majority of those with SARS-CoV-2 infection do eventually develop symptoms, an appreciable prevalence of asymptomatic – rather than pre-symptomatic – infection has been described in hospital patients (12.4%)18, nursing home residents (3.9%)12 and healthcare workers (0.5%)8. The contribution that these individuals make to transmission is less clear. The absence of cough and coryza might limit infectivity. Alternatively, a period of viral shedding without the social distancing and reductions in numbers of close contacts, which would have otherwise been prompted by symptom onset, might result in a greater number of secondary infections. Data suggest that pre-symptomatic transmission probably plays a more important role than asymptomatic transmission16. However, until this is better understood it would be prudent to consider both asymptomatic and pre-symptomatic people with positive SARS-CoV-2 PCRs infectious, particularly given this group are initially clinically indistinguishable.\n\nClearly, the prevalence or prior probability of SARS-CoV-2 infection, among both individuals with and without symptoms, will vary over the course of the pandemic19 and with changes in control strategies.\n\n\nTest characteristics\n\nViral load in the upper airway, the site most readily sampled, is thought to peak around the time of symptom onset13 and to be correlated with symptom severity20. An early analysis suggested that the sensitivity of PCR-based tests decreases with time since symptom onset, with only 67% of nasal and 47% of throat specimens expected to be PCR positive ten days after symptom onset21. In a small series of well-characterised patients with mild disease, test sensitivity was reported to be only 40% five days after symptom onset22. Later analysis, incorporating some of the same data, estimated that the sensitivity of the test was 62% on the day of symptom onset, 80% on day three after symptom onset, then declining to 33% by day 16 post symptom onset23. The sensitivity of a SARS-CoV-2 PCR on the day prior to symptom onset was estimated to be between 6% and 73%, with the imprecision in the estimate reflecting the limited data available on pre-symptomatic patients23. Most of these studies did serial testing and required at least one positive PCR to consider a case confirmed – as a result they may have overestimated the sensitivity of the test. Some of the studies included in Kucirka et al.23 allowed probable cases, defined on clinical grounds, with a majority of these probable cases also having IgM or IgG antibodies against SARS-CoV-2.\n\nData on test specificity is less readily available. As well as the inherent performance of the test, specificity can be impacted by: clinicians swabbing the wrong patient or placing the wrong sticker on the specimen; RNA contamination at any stage in the process; and transcription errors when reporting the result. The extent to which these errors occur will be context specific and dependent on how robust local procedures are. Importantly, PCR based testing only gives a snapshot at one point in time. Individuals early in their infection may test negative then positive a few hours later23. As with all PCR based tests, poor sampling may result in false negatives.\n\nReadily available formulae24 enable the probability of both false negative and false positive tests to be estimated under a range of assumptions about sensitivity, specificity, and the prevalence of the condition in the population being tested.\n\n\nTesting symptomatic staff in institutional care settings\n\nA major concern during the COVID-19 pandemic has been health systems’ ability to cope with large numbers of acutely unwell individuals presenting to health facilities within a short period of time, overwhelming capacity. The availability of skilled clinicians is a major constraint on health system capacity and high rates of absenteeism have been reported at many centres25. Absent staff were either symptomatic, self isolating because a household contact was symptomatic, or not working as a result of medical conditions that would put them at risk of severe disease were they to acquire SARS-CoV-2. There has, thus, been great interest in SARS-CoV-2 PCR tests for symptomatic health and social care workers and their symptomatic household contacts.\n\nWhilst reducing absenteeism is clearly important, a key concern when testing symptomatic individuals is ensuring that false negative results don’t result in avoidable transmission of the virus among keyworkers and the vulnerable individuals they work with. Estimated false negative rates, under a range of plausible assumptions, are presented in Table 1.\n\nAll estimates assume a test specificity of 99.5%.\n\nAs others have also noted21,23,26 high rates of false negative tests are seen when the prevalence of infection is high, and when the sensitivity of the test is low. The proportion of health and social care workers who have positive test results should be monitored with these data, ideally, disaggregated by symptoms. Where this proportion is high, there will be an unacceptable false negative rate and institutions should consider asking symptomatic staff to self isolate regardless of their test result. Examples of such risk stratification have been published8 but, in our view, should be kept under review, given the false negative rate will vary with the prevalence of infection in the population. It may be prudent to only test key workers shortly after symptom onset, when the sensitivity of the test is highest.\n\nIt is biologically plausible that staff with SARS-CoV-2 infection may be less infectious if they have a negative PCR. We know of no good data to support such an assertion and would urge caution, as negative results can be a result of poor sampling or virus being less readily detected in the upper respiratory tract than in samples taken from the lower airways27.\n\nAs others have also argued3, we feel it likely that any gains in terms of reduced absenteeism as a result of a programme of testing symptomatic staff may be modest. Staff with symptoms may be too unwell to work and, regardless, should not be working whilst symptomatic given other respiratory viruses can also be transmitted to patients. Testing symptomatic household contacts may be more impactful with, potentially, a lower pre-test probability, allowing greater confidence in a negative result3.\n\n\nTesting asymptomatic staff\n\nArguably, a better use of tests, particularly if capacity is limited, would be systematic regular testing of asymptomatic staff, to drive down the prevalence of asymptomatic and pre-symptomatic infection within institutions caring for the vulnerable. One model suggests that weekly PCR testing of asymptomatic health and social care workers could reduce their contribution to incident infections by 25–33%, over and above any reductions as a result of them self-isolating at onset of symptoms3. Given virus is likely only detectable 1–2 days prior to symptom onset13,23, one might expect more frequent testing to result in greater declines in transmission, particularly if results could be fed back promptly. A second model gave similar results regards the impact of weekly testing and suggested that daily testing of hospital staff could reduce healthcare worker to healthcare worker transmission by 65% and healthcare worker to patient transmission by 14%7.\n\nWhilst, initially, a programme of testing asymptomatic staff might result in additional absenteeism, reductions in transmission may result in a favourable impact on staffing levels in the longer term7. False positive tests would cause unnecessary absence from work, but no direct harm to vulnerable individuals.\n\nThe main barrier to regular asymptomatic testing is test capacity. For example, there are 1.5 million people working in adult social care in England28. Pooling samples prior to testing could make regular systematic large scale testing of key workers feasible. This is discussed in detail below.\n\nThe impact of asymptomatic staff testing could be increased by calibrating test frequency to the proportion of staff and patients testing positive and by offering enhanced support with other aspects of IPC to facilities with higher than expected rates of positive tests – this approach has been described in the hospital setting8.\n\n\nTesting asymptomatic patients\n\nFollowing reports of high rates of asymptomatic infection in patients presenting to hospital with other issues18, many hospitals are now undertaking SARS-CoV-2 PCR tests on all new admissions, with the result used to distribute patients to ‘COVID areas’, ‘non COVID areas’ or side rooms. With potential for harm should people with SARS-CoV-2 infection and vulnerable people who have not yet been infected be placed in the same space, it is important to think critically about the positive and negative predictive value of the test in specific circumstances.\n\nPeople with a positive PCR result and symptoms consistent with COVID-19, as well as asymptomatic individuals with a negative PCR test, pose fewer challenges when drafting infection control guidelines. Note, however, that the latter group may subsequently become PCR positive, either a result of nosocomial infection or because they were in their incubation period and PCR negative at presentation.\n\nPeople with COVID-19 symptoms, or imaging and blood tests consistent with COVID, but a negative SARS-CoV-2 PCR are commonly seen in clinical practice29. Sometimes repeat PCR testing, particularly if a sample from the lower respiratory tract can be obtained, can confirm the diagnosis. Efforts should also be made to exclude alternative diagnoses, such as Pneumocystis jirovecii pneumonia, which can present in a similar manner.\n\nThe opposite phenomenon – a positive SARS-CoV-2 PCR in an asymptomatic person – can also occur. However, the possibility of this being an erroneous result is less well appreciated. In Table 2, are the probabilities of SARS-CoV-2 infection given a positive PCR that might be expected under various scenarios. False positive results will commonly be seen, particularly in settings and populations where the prevalence of infection is low.\n\nAll estimates assume a test specificity of 99.5%.\n\nIdeally, individuals with PCR test results that are not concordant with their symptoms would be cared for in side rooms. However, many institutions have a limited number of side rooms, meaning such a policy may not be sustainable. Requiring a second positive test in asymptomatic people with an initial positive PCR would seem prudent before moving them into a ‘COVID area’.\n\nWhere individuals’ infection status cannot be resolved and side rooms are not available, every effort should be taken to avoid placing individuals who would be particularly at risk of severe disease should they newly acquire infection in a space with individuals who may be infectious. Such a triage approach has been piloted at one hospital – in this case with the triage decision taken prior to the PCR result being available29. The performance of any such algorithm will vary with the background prevalence of SARS-CoV-2 infection. If vulnerable and potentially uninfected individuals must be moved out of side rooms, impeccable infection control measures should be adopted in the spaces to which they are moved, perhaps supported by better staff-to-client ratios. If possible, such spaces should be closed to new admissions to prevent these individuals being exposed to new people who may be infectious.\n\n\nPooling specimens prior to analysis\n\nOne way that testing capacity might be expanded is by pooling specimens prior to analysis. A recent report suggests that pooling of samples either prior to or following RNA extraction can allow multiple samples to be tested in a single PCR reaction without significant loss of sensitivity30.\n\nThe authors reported a 1.24 increase in the PCR cycle threshold with every two-fold dilution of the sample – i.e. going from one to two, or two to four samples. In their hands, pooling 32 samples following RNA extraction reduced the sensitivity of the test by 10%. They suggest that some of this loss of sensitivity might be overcome by running the PCR for a few additional cycles. Any loss of specificity as a result of additional PCR cycles would not be expected to impact patient management, as individual samples from pools that tested positive would then be retested using one PCR reaction per sample.\n\nThe probability that, in any pool of specimens, at least one will test positive is one minus the probability that all will test negative. Here, the prevalence is the prevalence of positive tests, were each to be tested in a separate PCR run, rather than the true prevalence of infection.\n\nProbability ≥1 positive = 1 – (1 – prevalence) ^ number of tests\n\nThe expected number of pools that test positive would be the product of this probability, the number of pools, and the sensitivity of the pooled approach, as compared with one PCR per sample. The number of infections missed, as compared with a one PCR reaction per sample strategy, would approximate the product of the true prevalence of infection, the loss of sensitivity as compared with a one PCR reaction per sample strategy, and the number of individuals tested.\n\nThe most efficient pool size depends on the prevalence (Figure 1). The scenario illustrated in this figure assumes a health or social care workforce of 10,000 individuals and the number of PCR reactions calculated assumes individual PCRs are then performed on each sample in pools that test positive. Again, prevalence is the prevalence of positive tests, were each to be tested in a separate PCR run, rather than the true prevalence of infection.\n\nThe most efficient pool size is readily calculable, and the prevalence of positive tests should be monitored to ensure the optimal pool size is being used. At higher prevalence, larger pools are less efficient, as the high proportion of pools testing positive limits the number of tests saved.\n\n- Briefing for staff on the planned testing strategy\n\n- Sampling frame drawn up for each ward/facility, to include all medical, nursing, allied health professionals, and domestic staff with a regular presence in the institution\n\n- Collect mobile phone numbers for each staff member, and issue each with a patient number, if they do not already have one\n\n- Collate rotas and prepare two swabs for each staff member per week, pre-labelled and in lysis buffer (likely to equate to approximately alternate day testing, assuming some rest days)\n\n- Allocate these to packets that are delivered to wards/facilities ahead of time with one packet for each morning handover\n\n- After morning handover, self nose and throat swabs by outgoing night staff and incoming day staff\n\n- Testing register to include space to note whether any staff have left or joined – swabs for new staff could be added to subsequent test packets\n\n- Specimens collected after handover to allow RT-PCR for SARS-CoV-2 on a mid morning PCR run – this may need a dedicated courier collection\n\n- Positive results called out before 5pm, to ensure incoming night staff don’t come on shift (if unable to contact staff member, message to be left with nurse in charge)\n\n- Individual letters, emails or text messages to each staff member, containing test results, plus reliable and timely communication of positive results to local infection control and health protection teams\n\n- Clear messaging to ensure symptomatic staff do not wait for their test result before leaving work, and that individuals developing symptoms self isolate regardless of whether they have had a recent negative test\n\n- Dedicated mobile phone and email address for any queries\n\n\nOrganisational considerations for asymptomatic staff testing\n\nIn designing a testing strategy for asymptomatic staff, there will be trade offs. For example, between a one PCR reaction per sample strategy and a pooled testing strategy. The former would be expected to result in the fastest results if there was plenty of capacity, whereas the latter would make most efficient use of limited capacity but add a few hours to the turn around time.\n\nDuration of infectiousness prior to symptom onset is thought to be between 2–3 days13 with limited PCR sensitivity expected more than two days prior to symptom onset23. We would, therefore, advocate an asymptomatic staff testing strategy that prioritises frequent testing and prompt reporting of results over marginal improvements in test sensitivity.\n\nHaving nose and throat swabs taken by individuals trained to do this might result in better sampling, increasing test sensitivity, but would place limits on how quickly testing could be scaled up. Of note, 1533 swabs taken by symptomatic staff at a hospital in England were all positive for RNAaseP, a human nucleic acid used as a sample quality control. This does not mean that sampling was perfect, but suggests that the swabs did all make contact with some human mucosa31. In a smaller study, good agreement was seen between PCR results using self swabs and swabs taken by a trained professional32.\n\nThere is some data to suggest that saliva may represent a viable alternative upper respiratory tract sample to nose and throat swabs for SARS-CoV-2 PCR. Of the two studies published to date, one found saliva had slightly better sensitivity than nose and throat swabs33, and one found the opposite34. As one might expect, given saliva samples are easier to collect, greater consistency was seen between PCR results on repeat saliva samples than with repeat nose and throat swabs33. Where self-sampling is planned, laboratories may wish to validate PCR assays using saliva.\n\nA suggestion as to how a mass testing strategy for staff might be organised is outlined in Box 1. Other choices may be more appropriate, depending on local circumstances. For example, care homes may wish to include regular visitors in their sampling frame and to mandate a recent negative PCR prior to visiting. Facilities using agency staff may wish to mandate a recent negative PCR prior to any shifts. Testing intensity could be calibrated to local epidemiology and testing capacity8, with a move to daily testing when capacity is available or if high rates of asymptomatic infection or disease are seen within facilities. Testing programmes should be developed in close collaboration with clinicians, infection control teams, health protection teams and the workers that are to be tested.\n\nDuration of self-isolation should be compliant with local guidelines, which are likely to evolve as our understanding of duration of infectiousness changes. Unless staff are immunocompromised, mandating negative PCRs before return to work would, in our view, be over cautious given PCR tests may detect non viable virus. Immunocompromised people should probably not be undertaking patient facing duties currently. In the absence of good data on duration of immunity following natural infection, pragmatic decisions may need to be taken locally about whether individuals who have had a positive SARS-CoV-2 PCR continue to be included in asymptomatic staff testing programmes.\n\n\nSerological tests\n\nIf and when we develop an understanding of the immunological correlates of protection against SARS-CoV-2 infection and or COVID-19, and have validated assays to measure this, they may compliment PCR based testing. For example, if we had a serological assay that could identify individuals with protective immunity to infection or disease, positive serology could allow someone to be safely moved out of a side room and into a ‘COVID area’. Such a test could also identify individuals that would no longer need to be included in asymptomatic staff testing programmes. For both of these purposes, a highly specific test would be needed.\n\n\nKey messages\n\nCorrect interpretation of SARS-CoV-2 PCR tests depends on the sensitivity of the test and prevalence of infection in the population being tested.\n\nTest sensitivity declines with time since onset of symptoms.\n\nThe prevalence of infection will vary between groups, with the symptoms people report, and over the course of the pandemic – monitoring the proportion of PCR tests that are positive can give an estimate of the prior probability of a positive test.\n\nWhere prevalence of infection is high, or more than three days have elapsed since symptom onset, a negative PCR may not reliably exclude SARS-CoV-2 infection and, in this situation, symptomatic individuals should isolate whatever their PCR result.\n\nWhere the prevalence of infection is low, false positive PCR tests may be commonly seen – it is prudent to obtain a second positive test before placing vulnerable individuals with a single positive test in a space containing other people with SARS-CoV-2 infection.\n\nUsing test capacity to reduce the prevalence of SARS-CoV-2 infection in institutions accommodating vulnerable individual should be a priority, with models predicting a substantial impact on transmission.\n\nA significant proportion of SARS-CoV-2 transmission occurs prior to symptom onset and there is a window of 1–2 days prior to symptom onset when a PCR test might be positive – regular testing of asymptomatic individuals and the prompt reporting of results are therefore needed.\n\nTest capacity can be substantially increased by pooling samples prior to testing – the most efficient pool size will depend on the prevalence of infection and is readily calculated.\n\nPhysical distancing and meticulous infection prevention and control will remain essential in institutions caring for vulnerable people.\n\n\nData availability\n\nNo data are associated with this article.", "appendix": "References\n\nWynants L, Van Calster B, Collins GS, et al.: Prediction models for diagnosis and prognosis of covid-19 infection: Systematic review and critical appraisal. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYelin I, Aharony N, Tamar ES, et al.: Evaluation of COVID-19 RT-qPCR test in multi-sample pools Idan. Clin Infact Dis. 2020; ciaa531. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKeeley AJ, Evans C, Colton H, et al.: Roll-out of SARS-CoV-2 testing for healthcare workers at a large NHS Foundation Trust in the United. Euro Surveill. 2020; 25(14): 2000433. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWehrhahn MC, Robson J, Brown S, et al.: Self-collection: an appropriate alternative during the SARS-CoV-2 pandemic. J Clin Virol. 2020. 128: 104417. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWyllie AL, Fournier J, Casanovas-Massana A, et al.: Saliva is more sensitive for SARS-CoV-2 detection in COVID-19 patients than nasopharyngeal swabs. medRxiv. 2020; 2020.04.16.20067835. Publisher Full Text\n\nWilliams E, Bond K, Zhang B, et al.: Saliva as a non-invasive specimen for detection of SARS-CoV-2. J Clin Microbiol. 2020; JCM.00776-20. PubMed Abstract \| Publisher Full Text" }	[ { "id": "66235", "date": "20 Jul 2020", "name": "Charles Agoti", "expertise": [ "Reviewer Expertise Molecular diagnostics/viral genomics/Respiratory/Enteric virus transmission in community settings." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nYates et al. vividly discuss utility and inherent limitations of current PCR testing of SARS-CoV-2 in institutions caring for the elderly and vulnerable adults. They begin by summarizing the SARS-CoV-2 epidemiology in this key population. The laboratory testing practical considerations and implications they highlight are very useful for both their specific target audience and the general scientific community to keep vulnerable populations safe. The messages of importance of quick sample-to-answer turnaround time, frequent testing to identify asymptomatic and pre-symptomatic infections, that PCR is not 100% accurate and that testing resources can be conserved by pooled testing are very important in these times. They provide suggestions on optimizing staff testing in care homes to monitor any SARS-CoV-2 infection introduction using available resources sparingly. I have three minor suggestions for the authors to consider.\nComment on if PCR cycle threshold (Ct) value is a useful indicator in evaluating the infectiousness of both staff and those under care and how this can be incorporated in making decisions in a care facility and in recognizing potential false positives?\n\nComment on whether sensitivity and specificity differ between PCR assays and how this can impact SARS-CoV-2 results in care homes. Any recommendations here? There are hundreds of commercial PCR assays available for SARS-CoV-2 testing. Some of the available PCR test check for multiple SARS-CoV-2 genes concurrently and include an internal control.\n\nComment on how/whether PCR tests can be used to identify contamination in care homes e.g. environmental swabbing, wastewater/sewage monitoring. Is this a viable strategy to monitor introduction or ongoing shedding/transmission?\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": "69772", "date": "11 Sep 2020", "name": "Sheila Lumley", "expertise": [ "Reviewer Expertise COVID: Asymptomatic healthcare worker surveillance/testing/contact tracing", "laboratory testing", "assay evaluation", "infection prevention and control" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nYates et al. discuss the current and highly topical issue of infection prevention control in institutional care facilities, focusing on the benefits of high frequency surveillance of asymptomatic and pre-symptomatic individuals, on absenteeism and infection prevention control. The article covers a range of important considerations, including sampling strategies (self-swabbing and saliva collection), testing strategies in particular pooling of samples, result interpretation given different population prevalence, and organisational considerations for implementing such a strategy. This article discusses how the pooling of samples might overcome the current capacity issues when implementing a high frequency mass screening strategy, given current constraints with NHS testing capacity to deliver screening programmes for large numbers of staff or patients.\n\nA number of papers on screening programmes in healthcare workers show high rates of nosocomial acquisition in staff who were ‘COVID-facing’. Much of this occurred in the early part of the pandemic in the UK before the introduction of universal PPE, with individuals looking after elderly patients or those with atypical COVID-19 presentations in the early part of the pandemic, leading to mode of acquisition in line with the reference quoted (89% of infections in healthcare workers may be a result of nosocomial transmission). However, following the introduction of universal PPE, the literature also documents high rates of staff to staff transmission in non-patient facing areas likely due to a failure of staff to socially distance away from clinical areas. In a study of over 10000 healthcare workers in an acute Trust the greatest risk of staff infection was associated with having a Covid-19 infected contact in the household (eLife 2020;9:e60675 DOI: 10.7554/eLife.60675)1. Most Healthcare settings are now seeing lower rates of nosocomial and staff to staff transmission, given universal mask wearing in the UK. The authors’ intended audience is those designing testing strategies in health and social care settings, however, a number of the strategies discussed also have broader application for mass screening in lower prevalence settings such as workplaces and educational facilities. Expanding local/regional access to testing, testing capacity, improving turnaround time, maintaining analytic performance appropriate for the setting and population prevalence will contribute importantly to the current national effort to curb the pandemic.\n\nComments to address: Turnaround times – a key factor in high frequency testing, including pre-shift testing, is short turnaround times. How does a pooling strategy effect this – it seems unlikely that the necessary turnaround times (i.e. results before returning for the next shift) could be met if samples are being analysed in a laboratory setting and repeat testing of individual samples from a positive pool is required. See: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325181/.2 Other assay formats could be considered such as point-of-care nucleic acid LAMP and rapid antigen tests – but given reduced sensitivity in comparison with laboratory based PCR assays modelling with respect to population prevalence would need to be carefully considered.\n\nReferencing: The COVID literature is expanding at an exponential rate. As with much of current COVID literature, many papers already exist on similar topics, the context of this opinion article within the current literature isn’t clear in places, with some citations lacking. This article would benefit from a more thorough literature review in the following areas:\nSample pooling is already used in a number of settings, with CDC guidance on the topic (https://www.cdc.gov/coronavirus/2019-ncov/lab/pooling-procedures.html). The widespread use isn’t represented in this paper, with only 1 reference. There are a number of other papers discussing pooling samples, more accurate representation of current literature is needed.\n\nTesting of saliva – there are multiple recent papers on this topic.\n\nPlease clarify this paragraph: '…high rates of false negative tests are seen when the prevalence of infection is high, and when the sensitivity of the test is low. The proportion of health and social care workers who have positive test results should be monitored with these data, ideally, disaggregated by symptoms. Where this proportion is high, there will be an unacceptable false negative rate and institutions should consider asking symptomatic staff to self isolate regardless of their test result.’ – when what proportion is high? The proportion of symptomatic vs asymptomatic? Proportion testing positive?\n\nSerology: “Such a test could also identify individuals that would no longer need to be included in asymptomatic staff testing programmes” – this assumes that we know whether these individuals can be re-infected and still shed/transmit virus. The SIREN project (SARS-CoV-2 Immunity & REinfection EvaluatioN) includes those previously infected so we can gain a better understanding of what having evidence of previous exposure means with respect to re-infection.\n\nThe article would benefit from a discussion of the information technology constraints – these are huge. Patient/sample identification at the point of test, sample labelling with barcodes allowing easier run-through processes in diagnostic laboratories, IT to support pooling/separation of pools, result notification to allow responses on an individual level and for contact tracing.\n\nConclusion At the time of writing this review (September 2020) the UK government is promising mass coronavirus testing (‘operation Moonshot’) but the technology to deliver this does not yet exist. It is vitally important to consider how mass-surveillance testing could be rolled out in institutional care settings. The pace at which testing is required to move forwards is unprecedented compared with anything we have seen before, and this opinion piece with its careful consideration of testing utility at different levels of population prevalence and assay sensitivity will be a useful article.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/9-671
https://f1000research.com/articles/9-670/v1	02 Jul 20	{ "type": "Research Article", "title": "Analysis of GABRB3 gene mRNA expression and motor coordination after administration of valerian extracts (Valeriana officinalis) in BALB/c mice", "authors": [ "Erwin Mulyawan", "Muhammad Ramli Ahmad", "Andi Asadul Islam", "Muh. Nasrum Massi", "Mochammad Hatta", "Syafri Kamsul Arif", "Muhammad Ramli Ahmad", "Andi Asadul Islam", "Muh. Nasrum Massi", "Mochammad Hatta", "Syafri Kamsul Arif" ], "abstract": "Background: Valeriana officinalis has often been used to treat sleep disorders as a traditional medicine for 2000 years. The sedative effect of valerian extract is facilitated by the GABAA receptor β3 subunit. The aim of this study is to determine the effect of valerian extract on GABRB3 gene mRNA expression and sedative effect in BALB/c mice. Methods: This is an experimental preclinical study using a posttest-only control group design. A total of 20 BALB/c mice were randomly allocated into four groups consisting of five mice each. Group I was given 5 ml of Aqua Dest (distilled water), group II was given 0.025 mg/10 g of diazepam, group III was given 2.5 mg/10 g of valerian extract, and group IV was given 5 mg/10 g of valerian extract. The drugs were administrated for seven days through a gastric gavage. The rotarod test was performed on the seventh day. A blood sample was taken on the first day before drug administration and after the rotarod test on the seventh day to be analyzed using quantitative real-time PCR. Results: GABRB3 gene mRNA expression showed a significant increase in groups II, III, and IV (p <0.0001). There was significant difference between group III and IV. The examination of motor coordination (rotarod test) showed a significant difference (p <0.05) between group I and group II, between group I and group III, and between group I and group IV. There was no significant difference between group II and both groups III and IV. Conclusions: GABRB3 gene mRNA expression was significantly increased after the administration of valerian extract. Based on the rotarod test, valerian extract and diazepam had a clinically similar sedation effect. A higher dose of valerian extract does not yield a higher level of GABRB3 gene mRNA expression nor sedative effects.", "keywords": [ "Valeriana officinalis", "GABRB3 genes", "sedative", "real-time polymerase chain reaction", "rotarod", "BALB/c" ], "content": "Introduction\n\nGABRB3 is the gene that encodes the GABAA receptor β3 subunit, which is one of the chloride sub-channels of the gamma aminobutyric acid (GABA) receptor in humans. The protein encoded by the GABRB3 gene is one of 13 sub-units of the multiple chloride canal sub-units that act as GABA receptors. GABRB3 is a member of the GABAA receptor family genes and has a heterometric structure of pentameric ion channel ligand channels1–3. GABA is a very important inhibitory neurotransmitter of the central nervous system. GABA has an important role in reducing the excitation of neurons by inhibiting the transmission of nerve impulses in the brain and also plays a role in the regulation of muscle tone. GABA is classified as an amino acid because it has an amine group4. GABA receptors are the target workplace of GABA. GABAA is one of the GABA receptors that works in 40% of the mammalian central nervous system as a major inhibitory neurotransmitter. GABAA receptors are the site of action of a variety pharmacological drugs including barbiturates, benzodiazepines and ethanol. The majority of gene coding in the GABAA receptor sub-units is arranged into four clusters on chromosomes 4, 5, 15 and X in the human genome. The cluster of genes is assumed to contribute to the expression of genes. There are two clusters that have three genes, namely α5, β3 and γ3 on chromosome 15. The similarity of all clusters is that there is a β subunit that shows the orientation of the reverse transcription process5–7.\n\nSleep is a vital physiological phenomenon, which maintains the balance of human body regulation. Insomnia is a temporary or persistent difficulty in starting or maintaining sleep. Insomnia should be treated optimally, for it decreases the quality of life, stamina, productivity, and even triggers mental illnesses. The impact of insomnia cannot be underestimated, because it can lead to more serious conditions and endanger health and safety. Therefore, every insomniac needs to find the right solution8–12. Diazepam is one of the oldest and most commonly prescribed drugs for treating sleep disorders and belongs to the benzodiazepines class of drugs. Benzodiazepines exert their sedative-hypnotic properties by increasing or potentiating the binding of major inhibitory neurotransmitter GABA at the GABAA receptor13. These receptors are found in the central nervous system and consist of five subunits of proteins: two alpha sub-units, two beta sub-units, and one gamma sub-unit. Under normal conditions, GABA binds to alpha subunits at the GABAA receptor and facilitates the entry of negatively charged chloride ions into neurons. Benzodiazepines bind allosterically to the gamma subunit of the GABAA receptor and increase GABA binding to alpha subunits. This results in more chloride ions being diffused into the neurons and hyperpolarization ensues, thus making neurons less responsive to excitatory postsynaptic potential stimulation. In other words, benzodiazepines suppress the central nervous system, hence the sedative effects. The use of benzodiazepines is countered by several adverse effects, such as daytime drowsiness and dizziness or lightheadedness14–17.\n\nAccording to the World Health Organization (WHO), up to 80% of the world's population uses natural herbal medicines18. The valerian plant (Valeriana officinalis) is often used to treat sleep disorders. The use of valerian to reduce tremors, depression, treat headaches, and as antispasmodics has also been reported. The three main chemical ingredients of this plant include essential oils (valerenic acid), valepotriates, and alkaloids. Valerian extract contains GABA, which works by depressing the central nervous system. Valerian's mechanism of action is similar to benzodiazepines, which increases the number of chloride ions that enter the neurons. One of the ingredients of valerian is glutamine, which has the ability to cross the blood-brain barrier and will then be converted into GABA. Valerenic acid inhibits the breakdown of enzymatic GABA, causing the amount of GABA in the synaptic space to remain high. Valerian works by inhibiting GABA reuptake, so that the concentration of GABA in the synaptic cleft remains high, which will induce sedation19–23.\n\nHowever, unlike benzodiazepines, valerian binds to the beta sub-unit of the GABAA receptor. Valerian also inhibits GABA metabolism, which increases the abundance of GABA and prolongs the effects of hyperpolarization. The sedative effect of valerian extract is facilitated by GABAA receptor sub-unit β3. A previous study has found that consumption of 400–450 mg valerian extract before bedtime can improve sleep latency and sleep quality. Administration of 2000 mg/kg valerian liquid suspension daily for seven days in mice resulted in not only sedation, but also hyperthermia, defecation, and reflex disorder. The available evidence suggests that valerian might improve sleep quality without producing side effects24–29.\n\nTo the best of the author’s knowledge, there have been no published studies at the level of gene expression yet. We aimed to evaluate the level of GABRB3 gene mRNA expression and sedative effect after administration of valerian extract. We hypothesized that the administration of valerian extract results in increased GABRB3 gene mRNA expression and sedation in BALB/c mice. BALB/c mice have the characteristics of easy breeding and minimal weight variations between males and females. Therefore, it is the one of the most extensively used strains in experimental studies, particularly in neurobiology and neuroscience research. BALB/c mice were also used in our reference study regarding the use of valerian extract30.\n\n\nMethods\n\nAnimals were treated according to the Ethical Principles and Guidelines for Scientific Experiments on Animals (2005) by the Swiss Academy of Medical Sciences and ARRIVE guidelines for animal studies. All experimental protocols employed in this study were approved by the Medical Research Ethics Committee of Hasanuddin University Makassar, Indonesia (903/H4.8.4.5.31/PP36-KOMTEK/2017). Principles for the ethical treatment of animals in research environments are described (Government Regulation of Republic of Indonesia No. 95 2012). Specific and detailed directions for the care and use of animals in biomedical research are available in the guidelines, developed in 2011 by Indonesia Health Research Ethics Committee of the Ministry of Health (Indonesia National Guidelines on Health Research Ethics 2011)31.\n\nThis study was conducted at the Laboratory of Molecular Biology, Faculty of Medicine, Hasanuddin University, Makassar. This study was an experimental laboratory study with BALB/c mice as an animal model, using a posttest-only control group design.\n\nThis is an experimental study using 28 healthy adult male BALB/c mice that were eight weeks old, weighed approximately 25–35 grams, with no anatomical abnormality, that had never been used in experimental studies before. Mice were obtained from the maintenance and development unit of the experimental animal laboratory of Molecular Biology Faculty of Medicine, Hasanuddin University, Makassar, Indonesia. Out of 28 mice, 20 mice that could last ≥180 seconds in at least two of the three trials were included. All mice were housed in standard polycarbonate cages (60 cm long × 30 cm wide × 30 cm high) with rice husks, with each cage consisting of five mice. The animal housing kept was at 25±2°C under a 12-hour light/dark cycle. Mice were fed on standard diet in the form of mouse pellets (food made by Japfa Comfeed Indonesia Inc) and given tap water ad libitum for a period of seven days during this study. We assessed the clinical status of the mice every day during routine checks to prevent any clinical changes in the laboratory mice. No mice were found dead or having decreased appetite/growth during the study period. The mice were handled in accordance with the regulations set by the Animal Care and Use Committee at Indonesian National Guidelines on Health Research Ethics31.\n\nValerian extracts used in this study were Blackmores Valerian Forte, a standardized herbal pill equivalent to 2 g (2000 mg) of Valeriana officinalis dry root or rhizome. The extract was diluted with Aqua Dest (distilled water) to 2 mg/0.1 mL. The drugs were administered via gastric gavage with 1 ml syringe. The dose was determined with the dose used in humans as a reference; the maximum tolerated dose in humans is 81.2 mg/kg as determined by Kakeshi (2014)32, or 51 mg/kg by Al-Majed (2006)29. The Food and Drugs Administration (FDA) recommends converting the doses from human to mice by using 12.3 as a conversion factor, which led to the final dose of 2.5 mg/10 g and 5 mg/10 g as the dose for mice based on the average weight of 20 grams. Meanwhile, the dose of diazepam was adjusted from the human daily dose of 0.2 mg/kg, leading to the final dose of 2.5 mg/kg or 0.025 mg/10 g33,34.\n\nThe rotarod test is one of the most commonly used methods to assess motor coordination and balance in mice. It has also been used to assess sedation level effectively in previous studies. The latency of falling from the rod at a constant speed is sensitive to the presence of a biological change (i.e. sedation) that affects motor coordination and balance35,36. A rotarod consists of rods with multiple pointed edges along the seam, with a rod diameter of 3-7 cm, speed control, and a lever that halts the timer after the mice fell from the rod37. The mice underwent a \"trial\" using the rotarod machine for 300 seconds, at a speed of 20 rotations per minute (rpm), repeated three times with intervals of 10–15 minutes. The trial was conducted 24 hours before the study began. All mice underwent rotarod machine \"training\" for 60 seconds, at a speed of 20 rpm, repeated three times with five minute intervals, for seven days, 30 minutes before drug administration. On the seventh day, 30 minutes after drug administration, mice were placed on the rotarod machine for 300 seconds, speed of 20 rpm, repeated five times with 10–15 minute intervals. The timer starts when the rotary engine is running. The timer stops when the mouse falls from the rod or until 300 seconds is reached. The time taken to fall from the rod was recorded.\n\nAfter two-weeks acclimatization the mice were randomly divided into four groups (group I Aqua Dest, group 2 diazepam, group 3 valerian 2.5 mg and group IV valerian 5 mg) consisting of five animals each using the GraphPad calculator tool to prevent any bias. The sample size in this study refers to the minimum sample size according to the WHO for research on traditional short-term treatments using animal models, which is five animals per group and Federer’s formula38. The intervention group, group I, was given 5 ml of Aqua Dest; group II was given 0.025 mg/10 g of diazepam diluted with distilled water to 0.5 mg/0.1 mL; group III was given 2.5 mg/10 g valerian extract diluted with distilled water to 2 mg/0.1 mL; and group IV was given 5 mg/10 g valerian extract diluted with distilled water to 2 mg/0.1 mL. All interventions were administered once daily every morning at 08:00 am for seven days through a gastric gavage in the animal laboratory. All mice were fasted for three hours before drug administration. The body weight of mice was also recorded before drug administration. On the seventh day, 30 minutes after drug administration, mice were placed on the rotarod machine. The time taken to fall from the rod was recorded. Mice were returned to the cage to rest during the intervals. A blood sample of 0.2 ml was taken on the first day before drug administration and after the rotarod test on the seventh day to be analyzed using quantitative real-time PCR (Bio-Rad CFX Connect, USA). The blood was taken from the tail vein without anesthesia. Efforts were made to minimize suffering, such as limiting the frequency of blood sampling, providing adequate space for living and performing no other experimentation or pain inducing procedures.\n\nThree important steps in this method are lysing, binding, and washing according to the Boom methods39. A 100 µg/µl of blood volume sample was dissolved into 900 µl of L6 solution, which consists of 120 g of guanidium thyocyanate (GuSCN) (Fluka Chemie AG, Buchs, Switzerland, cat no. 50990) in 100 ml of 0.1 M Tris HCl, pH 6.4, 22 ml 0.2 M ethylenediaminetetraacetate (EDTA) pH 8.0 and 2.6 g Triton X-100 (Packard, Instrumens) with a final concentration of 50 mM Tris HCl, 5 M GuSCN, 20 mM EDTA, and 0.1% Triton X-100. The solution was centrifuged at 12,000 rpm. After the lysing process, 20 μl of diatom suspension consisting of 50 ml H2O and 500 μl of 32% (w/v) Celite (diatom) (Jansen Chimica, Beerse, Belgium, 10.846.79) was added. Around 20 μl of this diatom suspension can bind to 10 μg RNA/tissue/blood DNA. Then, a vortex was performed, followed by centrifugation in a 1.5 ml Eppendorf tube with a speed of 12,000 rpm for 15 minutes. The supernatant was removed and the sediment was washed with 1 ml L2 solution, consisting of 120 g GuSCN in 100 ml of 0.1 M Tris-HCl, pH 6.4. The mixture was vortexed and centrifuged at 12,000 rpm for 15 minutes, and washed two times with an L2 solution, and washed again with 1 ml of 70% ethanol twice and with 1 ml of acetone. The solution was then heated in water at a temperature of 56°C for 10 minutes and 60 μl of TE solution, consisting of 1 mM EDTA in 10 mM Tris HCL pH 8.0, was added. The solution was then vortexed and centrifuged at 12,000 rpm for 30 seconds and incubated in the oven for 10 minutes at a temperature of 56°C. Following the incubation, the sample was vortexed and centrifuged again at 12,000 rpm for 30 seconds. The supernatant was obtained by nucleotide extraction and stored at -80°C before PCR analysis.\n\nSamples for GABRB3 gene mRNA measurements were collected from the tail vein (0.2 ml for each sample). Total RNA was isolated from venous blood using the Qiagen RNeasy Micro Kit (DNA Genotek, Qiagen, Cat No 74004) according to the manufacturer’s instructions. Complementary DNA synthesis was performed by using iScript™ Advanced cDNA Synthesis Kit for RT-qPCR (Bio-Rad, Hercules, CA, USA, Cat No 172503) according to the manufacturer’s instructions. The synthesized cDNA was diluted 10 times and used to determine GABAAR subunit mRNA levels. Following reverse transcription, quantitative RT-PCR (qRT-PCR) was performed in triplicate using the iQ SYBR Green Supermix (Bio-Rad, Cat No 170-888) PCR mix containing 100 mM KCl, 40 mM Tris-HCl pH 8.4, 0.4 mM of each dNTP, 50 U/mL DNA Polymerase (iTaq), 6 mM MgCl2, SYBR Green I, 20 nM fluorescein, and stabilizers. The reaction was performed using a thermocycler (Real-Time PCR detection system C1000/CFX96, Bio-Rad, Hercules, CA, USA) in a final volume of 25 μL (5 μL RNase-free H2O, 5 μL cDNA template, 2.5 μL of each primer, and 12.5 μL of 2x iQ SYBRTM Green Supermix) under the following PCR conditions: initial heating at 95°C for three minutes to denature the cDNA and activate the Taq DNA Polymerase, followed by 40 cycles consisting of denaturation at 95°C for 30 seconds, annealing at 60°C for 60 seconds, and extension at 72°C for two minutes. The reaction was then stopped with a final step at 72°C for 15 minutes. Qiagen QuantiTect Primer Assay systems with the following 10X primers were used: GABRB3 (product number 249900, NM_021912, final concentration 1X), forward (5’ GCTTCTTGGCTTCTCTGGTG -3’) and reverse (5’- AACGAGATGCCATTCACTCC -3’). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (product number 249900, NM_002046, final conc.1X) was used as an internal control for normalization, GAPDH primer, forward (5’ TCCAGGGTTGGTCTTAGTGG -3’) and reverse (5’- TTCCACCAGGCTCTCTGTCT -3’). Analysis was performed using CFX Manager software (Bio-Rad) for calculating gene expression in the analysis mode ΔΔC(t) according to the manufacturer’s instructions. All measurements were conducted as per manufacturer’s instructions and repeated three times to ensure the validity. Target mRNA expression was read as fold change40,41.\n\nStatistical analysis was performed using the IBM® SPSS® Statistics version 26.0 for Windows (SPSS Inc., Chicago, IL, U.S.A.). Data normality tests were performed using the Shapiro-Wilks statistical test. ANOVA and Kruskal Wallis tests were used to analyze numerical differences between the four groups. The paired t-test was used to analyze mean values before and after treatment between each group, while the independent t-test was used to analyze mean values before and after treatment between two different groups. The analyzed variables were mouse weight, GABRB3 gene mRNA expression, and rotarod test results. A p-value of <0.05 was considered significant.\n\n\nResults\n\nGABRB3 mRNA expression before and after treatment showed normal data distribution, with a p-value of >0.05 within each group (Table 1)42. The Shapiro-Wilks normality test was also performed on motor coordination functions (Table 2). In this study, motor coordination functions were tested with the rotarod test, which showed a mean of 183.71 seconds with a median of 215.9 seconds42. The normality test showed normal data distribution with p-value of >0.05 in each group.\n\nData were analyzed with the Shapiro-Wilks normality test; a p-value of >0.05 was considered as normal data distribution. The unit used to measure the expression was fold change.\n\nData were analyzed with the Shapiro-Wilks normality test; a p-value of >0.05 was considered as normal data distribution. The values indicate seconds before falling.\n\nThe Kruskal Wallis test (Table 3) showed that there was no significant difference between the weight of each mouse (p>0.05)42.\n\nData were analyzed by the Kruskal Walis test; a p-value >0.05 was considered significant.\n\nTable 4 shows the mean value of GABRB3 mRNA expression before and after treatment. In group I, there was no significant difference (p = 0.653). In group II, there was a significant increase (p <0.0001) from 5.88 to 8.064. The increase was most significant in group III (p <0.0001), from 6.321 to 12.796. In group IV, there was an increase from 6.028 to 9.778 (p <0.0001). The ANOVA test (Table 5) did not show any significant difference between each group before treatment (p = 0.562). However, there was a significant difference after treatment (p <0.0001). The independent t-test (Table 6) did not show any significant difference (p >0.05) between two groups before treatment. However, there were significant differences between two groups after treatment (p <0.0001) (Table 7).\n\nData were analyzed by the paired t-test; a p-value of <0.05 was considered significant. The unit used to measure the expression was fold change.\n\nData were analyzed with the ANOVA test; a p-value of <0.05 was considered significant. The unit used to measure the expression was fold change.\n\nData were analyzed with the independent t-test; a p-value of<0.05 was considered significant. The unit used to measure the expression was fold change.\n\nData were analyzed with the independent t-test; a p-value of<0.05 was considered significant. The unit used to measure the expression was fold change.\n\nTable 8 shows the comparison of rotarod test results after treatment between two groups using the independent t-test. There were significant differences (p<0.05) between group I (Aqua Dest) and group II (diazepam), between group I (Aqua Dest) and the first treatment group (valerian 2.5 mg/10 g), and between group I (Aqua Dest) and the second treatment group (valerian 5 mg/10 g). There were no significant differences between the positive control group and either treatment group (p >0.05).\n\nData were analyzed with the independent t-test; a p-value of <0.05 was considered significant. The values indicate seconds before falling.\n\n\nDiscussion\n\nSedation is defined as a condition of decreased awareness of the surrounding environment and reaction to external stimuli. The sedative effect of several active substances in valerian extract work synergistically. Valerenic acid inhibits the breakdown of GABA, which results in an abundance of GABA in the synaptic space. Valerian extract also contains GABA, which suppresses the central nervous system. Furthermore, glutamine, one of the valerian extract substances, can cross the blood-brain barrier and will be converted into GABA. Lastly, valerian also acts by blocking GABA reuptake28,43.\n\nGABRB3 is a heteromeric gene that encodes the GABAA receptor β3-subunit, a member of the ligand-gated ion channel family in humans. GABAA receptors are the major inhibitory transmitter receptors in the brain and the site of action of a variety of pharmacologically and clinically important drugs1–4. Meguro et al. (1997) have identified GABRB3 expression in mouse A9 hybrids that contained either a paternal or maternal human chromosome 1544.\n\nAdministration of diazepam and valerian extract in this study resulted in a significant increase in GABRB3 gene mRNA expression. The first treatment group and second treatment group had the highest motor coordination function impairment compared to other groups, although not significant. Thus, it can be interpreted that valerian extract exhibited a sedation effect on both the clinical and molecular levels. Based on the result of the rotarod test, there were significant differences between group I and the rest of the groups, while there were no significant differences between groups II, III, and IV. This result indicated that valerian extract and diazepam have clinically comparable sedation effects, as shown in reports by Khom et al.5 and Budzinski et al.45 Valerian has been shown to have a sedative effect and prolong sleep time in mice, as reported by Hendrik et al.46 and Fernandez et al.20 Regarding motoric coordination function, Khom et al. also reported that the administration of ≥10 mg/kg of valerian extract caused a significant reduction in locomotion in mice5. The mice in this study have homogeneous characteristics (no difference in mean weight), thus not presenting as a confounding factor or bias that might affect the study’s results.\n\nInterestingly, the mice in first treatment group (valerian 2.5 mg/10 g) had the most significant increase in the mean value of GABRB3 gene mRNA expression after treatment (up to two-fold), exceeding that of second group treatment (valerian 5 mg/10 g), which had a higher dose of valerian extract. Furthermore, the mice in first treatment group also demonstrated the shortest mean duration in the rotarod test. These results indicate that increasing the dose of valerian extract (2.5 mg to 5 mg) was not directly proportional to the increase in both GABRB3 gene mRNA expression and sedation.\n\nThis study has several limitations. First, there are various available formulations of valerian extract without a clear standard of processing method. Second, there are no previous studies capable of proving that valerian extract exerts its sedative effects by increasing the level of GABRB3 gene mRNA gene expression. Finally, this study does not perform a brain biopsy for determining the level of GABRB3 gene mRNA gene expression through immunohistochemistry tests. Further research is needed to establish the standardized dosage of valerian extract. More definitive studies to determine the correlation between the level of GABRB3 gene mRNA expression and sedation effect are also warranted.\n\n\nConclusion\n\nValerian extract exerts its sedative properties by binding to the GABAA receptor β3subunit. As the gene that encodes the GABAA receptor β3 sub-unit, GABRB3 gene mRNA expression was increased after the administration of valerian extract. Valerian extract had a clinically similar sedation effect to diazepam. However, a higher dose of valerian extract did not yield a higher level of GABRB3 gene mRNA expression or sedative effects.\n\n\nData availability\n\nOpen Science Framework: Effect of Valerian Extracts on GABRB3 Gene mRNA Expression and Motor Coordination in BALB/c mice. https://doi.org/10.17605/OSF.IO/CT52W42.\n\nThis project contains the following underlying data:\n\n- Mice_Body_Weight.xlsx\n\n- mRNA_expression_of_GABRB3.xlsx\n\n- Rotarod.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": "Acknowledgments\n\nWe thank Mr. Jokevin Prasetyadhi from the Faculty of Medicine, Hasanuddin University, Makassar, Indonesia, who provided medical writing editing.\n\n\nReferences\n\nPapandreou A, Mctague A, Trump N, et al.: GABRB3 mutations: A new and emerging cause of early infantile epileptic encephalopathy. Dev Med Child Neurol. 2016; 58(4): 416–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRichter L, de Graaf C, Sieghart W, et al.: Diazepam-bound GABAA receptor models identify new benzodiazepine binding-site ligands. Nat Chem Biol. 2012; 8(5): 455–464. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMamoru F, Masaaki T: GABA-driven excitatory neurotransmission: gene regulation by excitatory GABA and its possible role in the developing brain. Neurotransmiter. 2015; 2: e480. Publisher Full Text\n\nSpitzer N: How GABA generates depolarization. J Physiol. 2010; 588(Pt 5): 757–758. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhom S, Hintersteiner J, Luger D, et al.: Analysis of β-subunit-dependent GABAA receptor modulation and behavioral effects of valerenic acid derivatives. J Pharmacol Exp Ther. 2016; 357(3): 580–90. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCassandra LK: Gamma-Aminobutyric Acid Receptor Beta 3: GABRB3. OMIM. 2008. (Accessed April 1, 2020). 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJames ML, Selleri S, Kassiou M: Development of Ligands for the Peripheral Benzodiazepine Receptor. Curr Med Chem. 2006; 13(17): 1991–2001. PubMed Abstract \| Publisher Full Text\n\nEkor M: The growing use of herbal medicines: issues relating to adverse reactions and challenges in monitoring safety. Front Pharmacol. 2014; 4: 177. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeuschner J, Muller J, Rudmann M: Characterisation of the central nervous depressant activity of a commercially available Valerian root extract. Arzneimittelforschung. 1993; 43(6): 638–641. PubMed Abstract\n\nFernández S, Wasowski C, Paladini AC, et al.: Sedative and sleep-enhancing properties of linarin, a flavonoid-isolated from Valeriana officinalis. Pharmacol Biochem Behav. 2004; 77(2): 399–404. PubMed Abstract \| Publisher Full Text\n\nShinomiya K, Fujimura K, Kim Y, et al.: Effects of valerian extract on the sleep-wake cycle in sleep-disturbed rats. Acta Med Okayama. 2005; 59(3): 89–92. PubMed Abstract \| Publisher Full Text\n\nHattesohl M, Feistel B, Sievers H, et al.: Extracts of Valeriana officinalis L. s.l. show anxiolytic and antidepressant effects but neither sedative nor myorelaxant properties. Phytomedicine. 2008; 15(1–2): 2–15. PubMed Abstract \| Publisher Full Text\n\nSundaresan N, Kasthuri B, Kaliappan I: Valeriana Officinalis: A Review of Its Traditional Uses, Phytochemistry and Pharmacology. Asian J Pharm Clin Res. 2018; 11(1): 36–41. Publisher Full Text\n\nYoo HK, Chung S, Hong JP, et al.: Microsatellite marker in gamma - aminobutyric acid - A receptor beta 3 subunit gene and autism spectrum disorders in Korean trios. Yonsei Med J. 2009; 50(2): 304–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNeuhaus W, Trauner G, Gruber D, et al.: Transport of a GABAA receptor modulator and its derivatives from Valeriana officinalis L. s. I. across an in vitro cell culture model of the blood-brain barrier. Planta Med. 2008; 74(11): 1338–44. PubMed Abstract \| Publisher Full Text\n\nBenke D, Barberis A, Kopp S, et al.: GABAA receptors as in vivo substrate for the anxiolytic action of valerenic acid, a major constituent of valerian root extracts. Neuropharmacology. 2009; 56(1): 174–181. PubMed Abstract \| Publisher Full Text\n\nThorne Research: Valeriana officinalis. Altern Med Rev. 2004; 9(4): 438–41. Reference Source\n\nBent S, Padula A, Moore D, et al.: Valerian for Sleep: A Systematic Review and Meta-Analysis. Am J Med. 2006; 119(12): 1005–1012. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAl-Majed AA, Al-Yahya AA, Al-Bekairi AM, et al.: Studies on the cytological and biochemical effects of valerian in somatic and germ cells of Swiss albino mice. Food Chem Toxicol. 2006; 44(11): 1830–1837. PubMed Abstract \| Publisher Full Text\n\nJohnson M: Laboratory Mice and Rats. Mater Methods. 2012; 2: 113. Publisher Full Text\n\nNational Guidelines on Health Research Ethics: Health Research Ethics Committee, the Ministry of Health, Republic of Indonesia. Litbang Kementrian Kesehat. 2011. Reference Source\n\nKakeshi A, Kato A, Naomi I, et al.: Valerian Inhibits Rat Hepatocarcinogenesis by Activating GABA(A) Receptor-Mediated Signaling. PLoS One. 2014; 9(11): e113610. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNair AB, Jacob S: A simple practice guide for dose conversion between animals and human. J Basic Clin Pharma. 2016; 7(2): 27–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMulyawan E, Ahmad MR, Islam AA, et al.: Analysis of GABRB3 Protein Level After Administration of Valerian Extract (Valeriana officinalis) in BALB/c Mice. Pharmacogn J. 2020; 12(4): 821–827. Publisher Full Text\n\nStanley JL, Lincoln RJ, Brown TA, et al.: The mouse beam walking assay offers improved sensitivity over the mouse rotarod in determining motor coordination deficits induced by benzodiazepines. J Psychopharmacol. 2005; 19(3): 221–7. PubMed Abstract \| Publisher Full Text\n\nDunham NW, Miya TSA: Note on a Simple Apparatus for Detecting Neurological Deficit in Rats and Mice. College of Pharmacy, University of Nebraska, Lincoln 8. J Am Pharm Assoc Am Pharm Assoc. 1957; 46(3): 208–209. PubMed Abstract \| Publisher Full Text\n\nMann A: Techniques for Motor Assessment in Rodents. In: Movement Disorders. London: Elsevier, 2014.\n\nWorld Health Organization. Programme on Traditional Medicine: General guidelines for methodologies on research and evaluation of traditional medicine. World Health Organization, 2000. Reference Source\n\nBoom R, Sol CJ, Salimans MM, et al.: Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990; 28(3): 495–503. PubMed Abstract \| Free Full Text\n\nKuang J, Yan X, Genders AJ, et al.: An overview of technical considerations when using quantitative real-time PCR analysis of gene expression in human exercise research. PLoS One. 2018; 13(5): e0196438. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFollesa P, Floris G, Asuni GP, et al.: Chronic Intermittent Ethanol Regulates Hippocampal GABA(A) Receptor Delta Subunit Gene Expression. Front Cell Neurosci. 2015; 9: 445. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMulyawan E: Effect of Valerian Extracts on GABRB3 Gene mRNA Expression and Motor Coordination in BALB/c mice. 2020. http://www.doi.org/10.17605/OSF.IO/CT52W\n\nSheta S: Procedural sedation analgesia. Saudi J Anaesth. 2010; 4(1): 11–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMeguro M, Mitsuya K, Sui H, et al.: Evidence for uniparental, paternal expression of the human GABAA receptor subunit genes, using microcell-mediated chromosome transfer. Hum Mol Genet. 1997; 6(12): 2127–2133. PubMed Abstract \| Publisher Full Text\n\nBudzinski JW, Foster BC, Vandenhoek S, et al.: An in vitro evaluation of human cytochrome P450 3A4 inhibition by selected commercial herbal extracts and tinctures. Phytomedicine. 2000; 7(4): 273–82. PubMed Abstract \| Publisher Full Text\n\nHendriks H, Bos R, Woerdenbag HJ, et al.: Central Nervous Depressant Activity of Valerenic Acid in the Mouse. Planta Med. 1985; 51(1): 28–31. PubMed Abstract \| Publisher Full Text" }	[ { "id": "128838", "date": "06 Apr 2022", "name": "Boyang Jason Wu", "expertise": [ "Reviewer Expertise Cancer Biology", "Neuropharmacology", "and Endocrinology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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, Mulyawan et al. determined the effect of valerian extracts on GABRB3 gene mRNA expression and motor coordination in mice. Their results showed that valerian extracts exert a comparable effect to diazepam by increased GABRB3 gene mRNA levels while decreased motor coordination in mice, suggesting a previously unrecognized sedation effect of valerian extracts. Overall, this manuscript is clearly written with interesting results presented and appropriate statistical analysis used. There are a few concerns for the authors to consider to improve the manuscript.\nGABRB3 expression was analyzed only at the mRNA level, so how would this change correlate to the protein-level or receptor activity change if any? Has such correlation been established or implicated for any genes encoding GABA receptor subunits in the literature? This should be at least discussed to complement the mRNA-level analysis.\n\nValerian extracts did not show a dose-dependent effect on both GABRB3 gene expression and motor coordination. The possible underlying explanations should be discussed. Also, have valerian extracts with the two doses used in this study showed any dose-dependent effect on other measures from the literature? How about time-dependent responses?\n\nIt would be clearer and more reader-friendly to present the data as bar charts including the distribution of all measurement points in individual groups.\n\nIs the work 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": [] }, { "id": "134839", "date": "05 May 2022", "name": "Margot Ernst", "expertise": [ "Reviewer Expertise Neuropharmacology", "GABA-A receptors" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe changes in blood mRNA levels of GABRB3 in mice after treatment with valerian extracts or diazepam, alongside reduced rotarod performance. The results are presented well and worthy of publication. The text requires some work in several respects:\nLanguage issues: The first sentence of the introduction needs to be reworded: the term \"chloride sub-channels\" is inappropriate. It can alternatively be stated that b3 is a subunit that contributes to pentamer formation.\n\nIUPHAR terminology violations: The use of subscripts to denote isoforms as in \"β3\" is inconsistent. All subscripts should be removed as per IUPHAR (e.g. line 1 of the introduction).\n\nContents: Abstract, second line: “The sedative effect of valerian extract is facilitated by the GABA-A R beta3 subunit” - this is certainly not the case. A single subunit rarely mediates a pharmacological effect. In the case of valerenic acid, a binding site has been proposed that overlaps with the site used by etomidate at the interface between beta and alpha subunits1.\n\nIn the same vein, in the introduction, the sentence “valerian binds to the beta subunit” needs to be corrected accordingly to reflect the contribution of alpha subunits to the binding site.\n\nThe authors equate reduced rotarod performance with sedation – it can reflect ataxia and/or muscle relaxation as well. Without an open field test, sedation cannot be assessed. The term “reduced rotarod performance” would thus be strongly preferred over “sedation”.\n\nStudy design and methods: Why do the authors assume that GABRB3 will be upregulated? According to the literature, the application of compounds that cause increased function often induces compensatory down-regulation. I suggest rewording the hypothesis. Why were blood mRNA levels examined rather than brain levels?\n\nData presentation: I agree with reviewer 1 that some graphical data presentation would be helpful.\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": [] } ]	1	https://f1000research.com/articles/9-670
https://f1000research.com/articles/9-669/v1	02 Jul 20	{ "type": "Case Report", "title": "Case Report: A rare case of a vanishing unilateral effusion", "authors": [ "Emily Hoodless", "Arvind Arumainathan", "Dennis Wat", "Arvind Arumainathan", "Dennis Wat" ], "abstract": "Pleural effusions rarely spontaneously resolve, and we document an instance where this phenomenon occurred. Here, we report a case of a 95-year old female who presented with a unilateral pleural effusion, diagnosed as secondary to a haematological malignancy [diffuse large B-cell lymphoma (DLBCL)] which resolved spontaneously. This is the first case to describe spontaneous remission in a primary cavitary DLBCL complicated by pleural effusion.", "keywords": [ "pleura", "lymphoma", "pleural effusion", "malignant", "neoplasm", "remission", "case report" ], "content": "Introduction\n\nPleural effusions are frequently encountered within the respiratory outpatient setting. There are a wide range of causes and potential interventions to be offered in order to diagnose and treat effusions. Pleural effusions rarely spontaneously resolve, and here we report an interesting case where this rare phenomenon occurred.\n\n\nPatient information\n\nA 95-year old Caucasian female was seen in July 2018 in a community respiratory clinic with a 4-month history of increasing breathlessness and a widespread vasculitic-looking rash. She had a background of high blood pressure and stable non-vasculitic chronic kidney disease. She lived alone and was fully independent with all activities of daily living.\n\n\nClinical findings\n\nThe clinical features on examination were in keeping with a left sided pleural effusion.\n\n\nTimeline\n\nThe patient was followed over the course of 6 months in 2018.\n\n\nDiagnostic assessment\n\nShe had a chest X-ray (Figure 1a) which showed a moderate left sided effusion, and subsequent computed tomography (CT) (Figure 1b) confirmed the presence of an effusion, along with mediastinal lymphadenopathy. An echocardiogram showed normal left ventricular function with moderate mitral stenosis and mild mitral regurgitation. A positron emission tomography (PET) CT scan did not suggest any size significant or fluorodeoxyglucose (FDG) avid hilar or mediastinal lymphadenopathy.\n\nClinical imaging of the patient (A) Chest radiograph showing left sided pleural effusion (B) computed tomography (CT) chest showing left sided pleural effusion (C) CT chest showing resolution of left sided pleural effusion.\n\nPleural aspiration was performed with 700 millilitres of yellow serous fluid obtained (exudative, protein - 47g/l, lactate dehydrogenase (LDH) - 3072U/l, negative microbiology culture). Other investigations demonstrated a negative vasculitis screen, serum LDH of 392U/l (<250U/l), and a small paraprotein was detected on immunofixation.\n\nFluid cytology revealed a diffuse population of intermediate to large sized lymphoid cells containing moderate amphophilic cytoplasm and round nucleus with fine chromatin and conspicuous nucleoli, some of them with plasmacytoid appearance. The lesional cells were of lymphoid B-cell lineage as evidenced by strong, diffuse expression of CD45, CD20 and PAX-5. A proportion of these cells co-expressed BCL-6 (90% of cells), BCL-2 (90%) and MUM1 (>80%), but not CD10, CD5, CD56, CD138, TdT, CD30, CD38, Cyclin-D1 or CD23. There was c-myc expression in approximately 80%. The proliferation fraction estimated by Ki-67 marker was high at approximately 80%. TTF-1 and MNF116 were negative. EBER (Epstein Barr virus-encoded small RNA) in situ hybridization for Epstein-Barr virus (EBV) was negative. The appearances were consistent with a high-grade, non-Hodgkin B-cell lymphoma, in keeping with a diffuse large B-cell lymphoma, not otherwise specified (DLBCL NOS). The immunohistochemical appearances were those of a non-germinal centre B-cell-like (non-GCB) subgroup.\n\n\nTherapeutic intervention, follow up and outcomes\n\nWhile awaiting treatment planning, repeat CT showed improvement in the effusion with resolution 6 months later; without any intervention (Figure 1c). Two years on, the patient remains symptom free, and in apparent remission.\n\n\nDiscussion\n\nPrimary effusion lymphoma (PEL) is a rare subtype of diffuse large B-cell lymphoma (DLBCL) and is universally associated with human herpes virus-8 (HHV-8) that involves body cavities and causes serous effusions without detectable masses or lymphadenopathy1–4. It also occurs in immunocompromised patients infected with human immunodeficiency virus (HIV) and Epstein-Barr virus (EBV)2,4,5. On the other hand, many cases of DLBCL with lymphomatous effusions on serosal surfaces, and no detectable mass lesion like PEL, have been reported. These cases were not regarded as cases of PEL, but proposed to represent a new entity, ‘PEL-like lymphoma (PEL-LL)’6. A single-centre evaluation of 185 consecutive patients with DLBCL presenting with pleural effusions confer an independent predictor of poor survival in Cox regression modelling (hazard ratio 1.9)7.\n\nThe frequency of spontaneous regression of cancer has been estimated to be about 1 case per 100,000 patients, and approximately 20 cases are reported each year8. Hypernephroma, melanoma, neuroblastoma, leukaemia, and non-Hodgkin lymphoma are the most commonly reported cancers exhibiting spontaneous regression.\n\nIn the case we present, there was spontaneous regression of the effusion, however, the aetiology underlying the development of spontaneous remission of malignancy remains unclear. Proposed mechanisms to explain this phenomenon include immunological, hormonal and genetic factors; concomitant infections; elimination of carcinogens; surgical trauma of the primary tumour and induction of differentiation9,10.\n\nIn this case, there were no obvious precipitating factors such as infections. The apparent absence of EBER expression argues against a lymphoma associated with immune senescence. There was no evidence of acquired immunodeficiency, or prior therapeutic interventions. The Ki-67 protein is a cellular marker for proliferation; in this case it was 80%, suggesting the presence of a rapidly proliferative neoplasm11. Taken into consideration previous reports associating primary cavitary DLBCL with a poor prognosis, it is noteworthy that our patient has remained in remission 2 years following her initial diagnosis, without therapeutic intervention. To our knowledge, this is the first case to describe spontaneous remission in a primary cavitary DLBCL complicated by pleural effusion.\n\nIn the case of unexplained pleural effusion, suspected to have an underlying neoplastic aetiology, clinicians should consider the possibility of haematological malignancy in the differential diagnoses, and investigate accordingly. They should also be aware that rarely, these have the potential to spontaneously resolve.\n\nOur patient was relieved to not have had to endure invasive therapies.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nCesarman E, Chang Y, Moore PS, et al.: Kaposi's sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas. N Engl J Med. 1995; 332(18): 1186–1191. PubMed Abstract \| Publisher Full Text\n\nKim KH, Lee JH, Jeong HC, et al.: A case of human herpes virus-8 unrelated primary effusion lymphoma-like lymphoma presented as pleural effusion. Tuberc Respir Dis (Seoul). 2012; 73(6): 336–341. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTerasaki Y, Yamamoto H, Kiyokawa H, et al.: Disappearance of malignant cells by effusion drainage alone in two patients with HHV-8-unrelated HIV-negative primary effusion lymphoma-like lymphoma. Int J Hematol. 2011; 94(3): 279–284. PubMed Abstract \| Publisher Full Text\n\nAdiguzel C, Bozkurt SU, Kaygusuz I, et al.: Human herpes virus 8-unrelated primary effusion lymphoma-like lymphoma: report of a rare case and review of the literature. APMIS. 2009; 117(3): 222–229. PubMed Abstract \| Publisher Full Text\n\nNakamura H, Tsuta K, Nakagawa T, et al.: Human herpes virus 8-unrelated primary effusion lymphoma-like lymphoma in the pericardium: a case with latency type III Epstein-Barr virus infection showing good prognosis without chemotherapy. Pathol Res Pract. 2015; 211(12): 1010–1013. PubMed Abstract \| Publisher Full Text\n\nAlexanian S, Said J, Lones M, et al.: KSHV/HHV8-negative effusion-based lymphoma, a distinct entity associated with fluid overload states. Am J Surg Pathol. 2013; 37(2): 241–249. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPorcel JM, Cuadrat I, García-Cerecedo T, et al.: Pleural Effusions in Diffuse Large B-Cell Lymphoma: Clinical and Prognostic Significance. Lung. 2019; 197(1): 47–51. PubMed Abstract \| Publisher Full Text\n\nThirukonda V, Petrich A, Parekh S: Classical Hodgkin Lymphoma and Spontaneous Regression. Clin Adv Hematol Oncol. 2012; 10(11): 765–766. PubMed Abstract\n\nPapac RJ: Spontaneous regression of cancer. Cancer Treat Rev. 1996; 22(6): 395–423. PubMed Abstract \| Publisher Full Text\n\nOno K, Kikuchi M, Funai N, et al.: Natural killing activity in patients with spontaneous regression of malignant lymphoma. J Clin Immunol. 1996; 16(6): 334–339. PubMed Abstract \| Publisher Full Text\n\nBroyde A, Boycov O, Strenov Y, et al.: Role and prognostic significance of the Ki-67 index in non-Hodgkin's lymphoma. AM J Haematol. 2009; 84(6): 338–43. PubMed Abstract \| Publisher Full Text" }	[ { "id": "121116", "date": "27 Jan 2022", "name": "Paul Stockhammer", "expertise": [ "Reviewer Expertise Medical Oncology - Thoracic Oncology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe an interesting case of an elderly female patient presenting with unilateral pleural effusion, who was found to have most likely primary effusion lymphoma-like lymphoma (PEL-LL), a rare entity of DLBCL confined to the serosal space. By undergoing diagnostic and therapeutic thoracentesis without any subsequent lymphoma-directed therapy, the patient went into apparent remission for at least two years. Overall, this report is of clinical relevance, given that it describes a rare subtype among the broad category of DLBCL with a rather unusual outcome. However, there are several aspects of this report that need to be addressed:\nThe authors categorized the patient’s lymphoma as DLBCL-NOS, but in the discussion they exclusively focus on PEL and the recently described PEL-LL. Based on immunotyping and clinical scenario, the patient should have most likely been subtyped as PEL-LL, and this should be also clearly stated in the results part. I was wondering whether the authors could provide status of HHV8, HIV and HCV in this patient (if tested, besides EBV), given that particularly HHV8 status is essential in differentiating PEL from PEL-LL. The authors should use uniform and clear nomenclature and should avoid the term primary cavitary DLBCL given that this would incorporate and not differentiate between PEL and PEL-LL.\n\nThe abstract is misleading and should more precisely state PEL-like lymphoma instead of the broad category of DLBCL, given that most people associate the term DLBCL with systemic lymphoma, and primary effusion lymphoma represents a very distinct subtype of DLBCL.\n\nCan the authors comment on the vasculitic-looking rash, and if there was a presumed relation to her lymphoma?\n\nCan the authors include a post-thoracentesis chest X-ray, given that the therapeutic thoracentesis most likely resulted in resolution of the moderate pleural effusion. Only showing the follow-up CT scan several months later can be misleading and misinterpreted as spontaneous resorption during that time.\n\nBy stating that the pleural effusion spontaneously resolved, the authors overstate their main findings. They identified a moderate pleural effusion, which on average contains about 680mL as per (PMID: 236328631). As stated by the authors, a thoracentesis with about 700mL fluid removal was performed, which should be considered as therapeutic intervention and would thus not mean “spontaneous resolution” but rather “no re-accumulation in pleural effusion after therapeutic thoracentesis was observed”. Interestingly, HHV8-unrelated PEL-LL with spontaneous regression of malignant cells without any additional treatment besides thoracentesis have been already reported by several groups (PMIDs: 233199972, 98501793, 170718114). Given those reports, the authors main statement of describing “the first case of spontaneous remission in primary cavitary DLBCL” as well as the articles title of “vanishing unilateral effusion” is not accurate and should be toned down. The authors should discuss those issues, and also discuss the potential mechanism of inducing cytogenic complete remission with sufficient pleural fluid drainage in patients with PEL-LL (PMID: 190755465).\n\nThe authors cited Porcel et al. with the statement that “pleural effusions associate with poor prognosis in patients with DLBCL”. However, in the cited report by Porcel et al., patients with systemic lymphomas with or without pleural effusions were analyzed and not patients with primary effusion lymphoma. As per above, PEL and PEL-LL represent completely different entities compared to systemic lymphomas, and thus the authors’ statement is misleading and should be rephrased or excluded.\n\nThe authors should cite and discuss the review by Wu et al (PMID: 238972646), who performed the so far largest literature review on HHV-8 unrelated PEL-LL cases. As indicated by Wu et al., there are some cases of patients undergoing pleural fluid drainage as the only therapy.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] } ]	1	https://f1000research.com/articles/9-669
https://f1000research.com/articles/9-663/v1	01 Jul 20	{ "type": "Research Article", "title": "In silico screening of known small molecules to bind ACE2 specific RBD on Spike glycoprotein of SARS-CoV-2 for repurposing against COVID-19", "authors": [ "Bharath BR", "Hrishikesh Damle", "Shiban Ganju", "Latha Damle", "Hrishikesh Damle", "Shiban Ganju", "Latha Damle" ], "abstract": "Background: Human coronavirus (SARS-CoV-2) is causing a pandemic with significant morbidity and mortality. As no effective novel drugs are available currently, drug repurposing is an alternative intervention strategy. Here we present an in silico drug repurposing study that implements successful concepts of computer-aided drug design (CADD) technology for repurposing known drugs to interfere with viral cellular entry via the spike glycoprotein (SARS-CoV-2-S), which mediates host cell entry via the hACE2 receptor. Methods: A total of 4015 known and approved small molecules were screened for interaction with SARS-CoV-2-S through docking studies and 15 lead molecules were shortlisted. Additionally, streptomycin, ciprofloxacin, and glycyrrhizic acid (GA) were selected based on their reported anti-viral activity, safety, availability and affordability. The 18 molecules were subjected to molecular dynamics (MD) simulation. Results: The MD simulation results indicate that GA of plant origin may be repurposed for SARS-CoV-2 intervention, pending further studies. Conclusions: Repurposing is a beneficial strategy for treating COVID-19 with existing drugs. It is aimed at using docking studies to screen molecules for clinical application and investigating their efficacy in inhibiting SARS-CoV-2-S. SARS-CoV-2-S is a key pathogenic protein that mediates pathogen-host interaction. Hence, the molecules screened for inhibitory properties against SARS-CoV-2-S can be clinically used to treat COVID-19 since the safety profile is already known.", "keywords": [ "SARS-CoV-2", "Pathogen host interaction", "SARS-CoV-2-Spike Glycoprotein", "ACE2", "Small Molecules" ], "content": "Introduction\n\nThe complete genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is 82% identical to SARS-CoV, both viruses share a common clade encompassing the genus Betacoronavirus as the root node1,2. Currently no novel antivirals exist that are effective against either of the viruses3–6.\n\nDrug repurposing is a commercially viable strategy, as it exploits existing drugs, thus significantly reducing the cost and time involved in developing effective therapeutics7–9. Experimental approaches, however, at pre-clinical and clinical stages for drug repurposing involve high cost and time10. Computational approaches can offer quick, considerable, and novel testable hypotheses for systematic drug repositioning8.\n\nCurrent drugs in different phases of clinical trials are being investigated for inhibitory activity against viral targets that play a significant role in the coronavirus infection lifecycle. The drug targets might be involved in entry into the host (e.g. umifenovir and chloroquine), replication (e.g. lopinavir/ritonavir), or RNA synthesis (e.g. remdesivir/favipiravir). Among these, targeting SARS-CoV-2 cellular entry via the spike glycoprotein (SARS-CoV-2-S) has emerged as the leading option for repurposing9. As SARS-CoV-2-S is a surface protein involved in adhesion/fusion and entry into host cells, it has been identified as a potential drug target for both biologics and small molecules10.\n\nThe entry of COVID-19 pathogen is mediated by the homotrimeric transmembrane protein SARS-CoV-2-S. It is comprised of two functional subunits, S1 and S2, which are non-covalently bound in the pre-fusion conformation. The S1 subunit interacts with the human ACE2 receptor through the receptor binding domain (RBD), while the S2 subunit is one of the components of viral envelope11–19.\n\nApart from interacting with the ACE2 receptor, the RBD site also contributes to the stabilisation of the prefusion state of the S2 subunit equipped with fusion machinery18–24. In CoVs, the S-protein is cleaved by host proteases at the S20 site located above the fusion peptide16,25. This activates the protein via extensive irreversible conformational changes11,16,17,23,26. It is well understood that the entry of CoV into the susceptible host is a complex process that requires the vigorous actions of receptor binding and proteolytic processing of the S-protein to promote fusion with the pathogen27.\n\nHence, the current study aims to predict and validate the structure of SARS-CoV-2-S protein using computer-aided homology modelling tools and screen a library of small molecules for their interaction with the SARS-CoV-2-S protein.\n\n\nMethods\n\nThe whole genome of SARS-CoV-2 (GenBank accession number: MT159721.1, length: 29882 bp) was retrieved from NCBI and used as a query to perform a sequence similarity search using NCBI-BLAST28. The BLAST search revealed 96.04%, 91.64% and 82.30% identity with Bat coronavirus RaTG13 (GenBank accession number: MN996532.1, length: 29855 bp), Pangolin coronavirus isolate MP789 (GenBank accession number: MT084071.1, length: 27213 bp) and SARS-CoV (GenBank accession number: JX163927, length: 29646bp), respectively.\n\nDrug target identification and validation were carried out using a network-based topological analysis method using the web-based application Pathogen-Host Interaction Search Tool (PHISTO) by setting pathogen type to virus, family to coronaviridae, species to SARS-Related coronavirus and strain to SARS-Cov. The node properties like the degree of connectivity (k) and betweenness centrality (BC) were assessed29,30. The statistical significance of k and BC values were assessed by the Fligner-Killeen (median) test.\n\nThe similarity between RBD domains of S-protein from SARS-CoV-2 (accession number QII57328, length:1273aa), SARS-CoV (accession number: AFR58728, length:1255aa) and RatG13 (accession number: QHR63300, length:1269aa) was evaluated by using the multiple sequence alignment (MSA) tool Clustal Omega from EMBL-EBI. Conservation in ACE2 receptor interaction was seen among all the three sequences aligned. This conservation aided in the active binding site prediction.\n\nThe protein-protein interaction between SARS-CoV-2-S and host ACE2 receptor complex was studied using the crystal structure from Protein Data Bank (PDB ID: 6CS2). The amino acids involved in the interaction were identified as ligand binding sites for inhibitor molecules.\n\nHomology modelling was performed with SWISS-MODEL for the protein sequence of SARS-CoV-2-S using the crystal structure of SARS-CoV-S and ACE2 complex (PDB ID: 6ACD) as a template. The modelled protein was validated for quality using a Ramachandran plot and prepared for molecular docking studies using the Protein Preparation Wizard feature of the Schrodinger Small Molecule Suite31. This analysis could also have been performed using open source software such as AutoDock32 or SwissDock33.\n\nThe modelled receptor was processed for docking studies by deleting crystallographic water molecules with less than three H-bonds. This could also be done manually by editing the .PDB file in a text editor. Next, hydrogen atoms corresponding to neutral pH were added in consideration of ionisation states of amino acids. Following this, coordinates for any missing side-chain atoms were added using Prime v4.0, Schrödinger 2019-234. Finally, the energy of the modelled structure was minimised using the OPLS_2005 force field35. This analysis could also have been performed using open source software such as AutoDock32.\n\nThe three-dimensional conformations of the 4015 small molecule drugs already in use to treat various diseases and as nutritional supplements were downloaded from the DrugCentral database and subjected to ligand minimisation using Ligprip (LigPrep, version 2.3, Schrödinger, LLC, New York, NY, 2009). This analysis could also have been performed using open source software such as AutoDock32. The compounds were minimised by assigning force field OPLS_2005 and stereoisomers were calculated after retaining specific chiralities. The absorption, distribution, metabolism and excretion (ADME) predictions were performed for all ligands using the QikProp package36. This analysis could also have been performed using open source software such as SWISS-ADME37.\n\nThe active site on the prepared receptor was defined around the selected residues (Arg426, Tyr436, Pro462, Thr486, Gly488, and Tyr491) with a 10Å radius. This generated a grid box measuring 20X20X20Å. The docking of small molecules over SARS-CoV-2-S was performed using Glide v7.8, This analysis could also have been performed using open source software such as AutoDock32. Schrödinger 2019-238 in different modes sequentially with defined and incremental precision, and computational time differences. The best-docked conformer with minimum Glide energy and E model energy was selected and lowest-energy docked complex of three known molecules streptomycin, ciprofloxacin, and glycyrrhizic acid (GA) in complex with SARS-CoV-2-S were selected for molecular dynamic simulations.\n\nThe MD39 of shortlisted complexes were studied using the OPLS_2005 force field40 in a plane TIP3P water model41. MD simulations were performed using Desmond version 4.242. This analysis could also have been performed using open source software such as GROMACS43. The system was built by dissolving the streptomycin/SARS-CoV-2-S, ciprofloxacin/SARS-CoV-2-S, and GA/SARS-CoV-2-S complexes in an orthorhombic box containing water molecules, allowing a buffer region of 10Å between atoms and box peripherals. The system was further minimised using the L-BFGS algorithm for a minimum of 10 steepest descent steps and a maximum of 2000 iterations until a gradient threshold of 25 kcal/mol/Å and convergence threshold of 1.0 kcal/mol/Å was reached. For short-range electrostatic interactions, the solid-phase microextraction44 method was employed at 1e-09 tolerance and 9Å cut-off radius. The built systems were gradually warmed up to 300K in the NPT ensemble with a time step of 2fs. A 100ns MD simulation in the NPT ensemble was performed using a Nose–Hoover thermostat40. Resulting root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values were analysed.\n\n\nResults and discussion\n\nThe complete genome of coronavirus SARS-CoV-2, Bat coronavirus RaTG13, Pangolin coronavirus isolate MP789, and SARS-CoV obtained as BLAST hits were aligned and a phylogenetic tree was constructed (Figure 1).\n\nThe MSA demonstrated the molecular similarities between the organisms. RaTG13 has been identified as a neighbour genome for SARS-CoV-2 and this justifies the hypothesis that the infection may be transmitted from bats. Meanwhile, the subsequent neighbours were Pangolin MP789 and SARS-CoV. This preliminary sequence alignment enabled the understanding of sequence similarities and evolutionary information, which is deeply fundamental to the process of drug discovery.\n\nThis figure was generated using EMBL-EBI Clustal Omega.\n\nA detailed investigation of the pathogen-host interactome can shed clear insights on the mechanism of viral infection and the pathology involved. Due to a lack of interaction data on SARS-CoV-2, the SARS-CoV proteome was considered and the SARS-CoV/human interactome was built by screening domain interactions between SARS-CoV/human protein-protein interactions, and then the network distribution, topological and functional analyses were performed (Figure 2). The circular shapes correspond to proteins (nodes) which are labelled by Uniprot_IDs and details about the nodes are listed in Table 1.\n\nAmong 14 proteins of SARS-CoV, the majority of SARS-CoV/human interaction involves five non-structural proteins (NS3B, NS6, NS7A, NS7B and NS8A with four, three, eight, three and one human proteins, respectively), three open reading frame (ORF) polyproteins (ORF9B, A7J8L3 and A7J8L2 with four, one and one human proteins, respectively), two replicase proteins (R1A and R1AB with two and one human proteins, respectively), the Membrane protein (VME1 with human IKKB), Envelope membrane protein (VEMP with human B2CL1), Nucleoprotein (NCAP with four human proteins) and Spike glycoprotein (SPIKE with human ACE2). With these observations, we determine the high specificity of Membrane, Envelope and Spike glycoprotein interactions with the host through specific entry points. Hence, these three SARS-CoV proteins can be a potential target to inhibit the pathogen-host interaction specifically, while other interactions are more versatile. To ensure the impact of inhibition of IKKB, B2CL1, and ACE2 mediated interaction, the landscape of the SARS-CoV/human interaction was further analysed for degree and betweenness centrality distributions of the host, as shown in Table 2.\n\nThe 28 blue nodes correspond to human proteins and 14 red nodes are SARS-CoV-2 proteins. The nodes are connected by 35 edges. This figure was generated using Pathogen-Host Interaction Search Tool (PHISTO).\n\nLUMIER, luminescence-based mammalian interactome mapping; ELISA, enzyme-linked immunosorbent assay.\n\nCumulative degree and betweenness centrality distributions.\n\nThe degree of connectivity estimates the number of directly connecting neighbours to a particular node, while betweenness centrality estimates the frequency of nodes occurring on the shortest paths in the context of other nodes. In the protein interactomes, a node with a high degree of connectivity is identified as hub protein and a node with maximum betweenness centrality is identified as bottleneck protein. In the current topological analysis, the node with the lowest degree of distribution 1 and betweenness centrality 0.0 was A2A3R6. However, the molecular function of A2A3R6 (Uniprot ID: A2A3R6) is not well understood in both human physiology or pathology. Hence, ACE2, with the degree of distribution 4 and betweenness centrality 2271, was the next most significant node, as shown in Figure 3, and it was identified as a key node or key player in the SARS-CoV/host interaction. Hence, the SARS-CoV-S interaction with host ACE2 was identified as a potential drug target. As information about the SARS-CoV-2/human interaction is not available, the SARS-CoV/human interaction data was used. We studied the similarity between SARS-CoV and SARS-CoV-2 by sequence analysis and RBD prediction.\n\nHost proteins that are targeted by SARS-CoV have an approximate degree and betweenness centrality. A) Degree distribution highlighting ACE2 position and B) betweenness centrality distribution highlighting ACE2 position. These findings are statistically significant by the Fligner-Killeen (median) test. This figure was generated using Pathogen-Host Interaction Search Tool (PHISTO).\n\nAs depicted in Figure 4, the alignment between the S-protein of SARS-CoV-2 and that of Bat coronavirus RaTG13 was closer than with the S-protein of SARS-CoV. The alignment at RBD site residues 317 to 569 was found to be more than 80% similar to SARS-CoV and RaTG13, particularly at major residues including Tyr436, Thr486, Gly488 and Tyr491 but excluding Arg426 and Pro462, as shown in Figure 5. Considering the evolution, the available elucidated structure of the SARS-CoV/ACE2 complex (PDB ID: 6CS2) was used as a template for homology modelling.\n\nThe residues involved in the interaction of SARS-CoV with ACE2 were predicted using the Prime module available in Schrodinger Small Molecule Suite and the major interactions are tabulated in Table 3 and shown in Figure 6. A very strong interaction was seen between the smallest amino acid, Gly488, with Lys353, Gly354, and Asp355. This interaction is facilitated by two features; one hydrogen bond and 94.9% buried solvent accessible surface area. Remaining residues also showed significantly strong interactions with ACE2. Hence, the same residues were made centric to generate the grid.\n\nThe residues with hydrogen bonding, better surface complementarity and buried SASA are tabulated and a few other weak interactions are ignored.\n\nHB, hydrogen bond; DS, disulfides, vWC, van der Wsals clash; SC, surface complementarity; B_SASA, buried solvent accessible surface area (SASA).\n\nThe residues highlighted in blue correspond to the B-Chain of SARS-CoV and residues in green correspond to the ACE2 D-Chain. This figure was generated using Schrodinger’s Maestro visualizer. As an alternative, the open source visualizer Python Molecule Viewer (PMV)48 could also be used.\n\nThe modelling of SARS-CoV-2 was performed using the crystal structure of SARS-CoV-S as a template, which was 97% identical to the query. The modelled protein shown in Figure 7A was validated for quality and preparedness. The Ramachandran plot generated using the protein preparation wizard confirmed the quality of modelled structure by plotting >95% residues in the allowed region, as shown in Figure 7B.\n\nA) The modelled structure of SARS-CoV-2 with Arg426, Tyr436, Pro462, Thr486, Gly488 and Tyr491 centric grid box generated for molecular docking, B) Ramachandran plot has >95% of amino acids plotted in the allowed region (red and yellow). This figure was generated using Schrodinger’s Maestro visualizer. As an alternative, the open source visualizer Python Molecule Viewer (PMV)48 could also be used.\n\nThe repurposing of small molecules as therapeutics to treat COVID-19 requires knowledge of the interaction of the therapeutic molecule with SARS-CoV-2-S. Initial high-throughput virtual screening suggested 142 molecules that exhibit reasonable interaction with SARS-CoV-2-S. Following this, the shortlisted molecules were docked in SP mode where the accuracy of prediction was improved. The docking in SP mode suggested 15 top molecules, listed in Table 4 as lead molecules. As hydroxychloroquine has been identified as a possible treatment for COVID-19, it was also subjected to subsequent docking in XP mode. All 15 molecules showed better interaction than hydroxychloroquine with SARS-CoV-2-S. The three molecules streptomycin, ciprofloxacin, and GA had low interaction penalties and displayed better interactions with the ACE2 binding site on the RBD of SARS-CoV-2-S, as shown in Figure 8A–C, respectively. The three molecules were selected based on their reported anti-viral activity, safety, availability, and affordability45–47.\n\nFor SARS-CoV-2-S, the Glide generated docking model showed that streptomycin could bind to SARS-CoV-2-S in a manner highly similar to the SARS-CoV-2-S and ACE2 interaction. The binding pocket of streptomycin was in the RBD site, which has been observed to be an acceptor for ACE2. Streptomycin was well-fitted with the shape of the pocket, as shown in Figure 8A and Figure 9A, with an XP score of -6.5, where it formed a total five hydrogen bonds, among which two hydrogen bonds were formed by donating electrons from N31 and N32 atoms to the Glu493 side-chain atoms. Simultaneously, two other hydrogen bonds were observed between the backbone atoms of Leu501 by receiving electrons from hydroxyl groups at the 5th and 6th carbon atoms of the S1 six-carbon ring of streptomycin. The remaining H-bonds were formed between the backbone atom of Ser503 and the hydroxyl group at the 6th carbon atom at the G3 group of streptomycin. However, the stability of the interaction cannot be pronounced without molecular dynamic simulations.\n\nA) Interaction of streptomycin facilitated by five H-bonds, shown as pink arrows. B) Interaction of ciprofloxicine facilitated by two H-bonds, shown as pink arrows. C) Interaction of glycyrrhizic acid, facilitated by give H-bonds, shown as pink arrows. This figure was generated using Schrodinger’s Maestro visualizer. As an alternative, the open source visualizer Python Molecule Viewer (PMV)48 could also be used.\n\nA) Interaction of streptomycin facilitated by five H-bonds. B) Interaction of ciprofloxicine facilitated by two H-bonds. C) Interaction of glycyrrhizic acid facilitated by five H-bonds. This figure was generated using Schrodinger’s Maestro visualizer. As an alternative, the open source visualizer Python Molecule Viewer (PMV)48 could also be used.\n\nThe docking model of ciprofloxacin illustrated its binding mode on the RBD site, which has been observed to be a key interference site for virus-host interaction. The ciprofloxacin fit with reasonable steric complementarity into the RBD pocket, as shown in Figure 8B and Figure 9B, with an XP score of -5.31. The interaction of ciprofloxacin with SARS-CoV-2-S was facilitated by two hydrogen bonds between Val492 and Phe499; each bond being formed by receiving and donating electrons from hydroxyl and ketone groups, respectively.\n\nThe docking model of GA illustrated its binding mode on the RBD site, which has been observed to be a key site for interference of the virus-host interaction. The GA fit with steric complementarity in the RBD pocket, as shown in Figure 8C and Figure 9C, with an XP score of -7.474. The docking of GA with SARS-CoV-2-S was facilitated by three hydrogen bonds with Leu464, Val492, and Glu493 by receiving electrons from the hydroxyl groups of GA. Additionally, the ketone group of GA formed a hydrogen bond, with the backbone atoms of Phe499 receiving the electrons.\n\nAs the SARS-CoV-2-S receptor has 1273aa, it requires enormous computational time to perform MD simulation for the whole range of protein, hence, we confined this study only to the RBD portion, ranging from 317th residue to 569th residue, for MD Simulation.\n\nRMSD analysis of protein-ligand complex. The RMSD can illustrate the average difference in the displacement of selected atoms in a particular frame compared to its reference frame. The plots in Figure 10 illustrate the evolution of a protein (left Y-axis) and ligand (right Y-axis) RMSD. Post simulation, the protein and ligand frames are initially aligned over the backbone atom coordinates of the reference frame, and then the RMSD is extrapolated. The information on protein-ligand RMSD can dissect and demonstrate the conformational differences that occurred throughout the simulation. The RMSD of between 1–3 Å is fairly acceptable for small, globular proteins. An RMSD exceeding this indicates a major conformational change during the simulation and pronounces the instability of the complex.\n\nA) streptomycin/SARS-CoV-2-S complex; B) ciprofloxacin SARS-CoV-2-S complex; C) glycyrrhizic acid/SARS-CoV-2-S complex. This figure was generated using Desmond version 4.242. This analysis could also have been performed using open source software such as GROMACS43.\n\nThe RMSD plot for the streptomycin/SARS-CoV-2-S complex, shown in Figure 10A, attained equilibrium at 5ns and thereafter showed stability with a maximum RMSD of 1 Å (peaks of 2.5 Å - 3.0 Å) up to 55ns. After 55ns a change in the equilibrium state was observed. However, the RMSD was within 1.5 Å, which is acceptable. Similarly, the streptomycin RMSD (right Y-axis) was observed to be significantly higher than the RMSD of the receptor at the RBD site. Thus, it is likely that streptomycin diffuses from its initial binding site after 48ns.\n\nThe RMSD plot for the ciprofloxacin/SARS-CoV-2-S complex, shown in Figure 10B, attained equilibrium at 2ns and thereafter showed stability with a maximum RMSD of 1.8 Å (peaks of 2.4 Å - 4.2 Å) up to 58ns. After 58ns a sudden change in equilibrium state was observed. However, the RMSD was within 2 Å, which is acceptable. On the other hand, the RMSD values for ciprofloxacin were observed to be significantly in alignment with the RMSD of SARS-CoV-2-S at the RBD site. Thus, it is likely that ciprofloxacin can retain its initial binding site up to 100ns.\n\nThe RMSD plot for the GA/SARS-CoV-2-S complex, shown in Figure 10C, attained equilibrium until 100ns. Compared to SARS-CoV-2-S complexes with streptomycin and ciprofloxacin, it was found that the SARS-CoV-2-S complex with GA was stable until the end of the simulation without any drift in equilibrium. On the other hand, the RMSD values for GA were observed to be significantly in alignment with the RMSD of the SARS-CoV-2-S RBD domain in almost all the frames. Hence, it is likely that it remains in its initial binding site up to 100ns. It is predicted to inhibit SARS-CoV-2-S at the RBD domain comparatively better than streptomycin and ciprofloxacin and for a longer duration, but its contact with key ligands has to be confirmed through RMSF and protein-ligand contact analysis.\n\nRMSF analysis. The RMSF helps characterise minute differences in the protein chain during the simulation. In RMSF plots, peaks correspond to the residues on the protein that fluctuate more during the course of a simulation. Usually, terminals and loop regions fluctuate more than other secondary structures like alpha-helices and beta-strands. The secondary structure of the RBD of SARS-CoV-2-S has the same secondary structural elements as the RBD from SARS-CoV, with 74% homologous residues. These residues are majorly formed of loops and are highly flexible. A unique Phe486 residue in the loop plays a key role in ACE2 interaction by occupying a deep hydrophobic pocket in ACE2. In the trimmed RBD structure this loop starts from 148th residue and ends at 172nd residue. Since the ligand-binding site is located in this loop region, a higher RMSD was noticed. In the RMSF plot for the RBD domain of the streptomycin/SARS-CoV-2-S complex, shown in Figure 11A, the RMSF at the loop region was 5.6Å with many ligand contacts (green-coloured vertical bars). This was on par with molecular docking interactions. In the RMSF plot for the RBD domain of the ciprofloxacin/SARS-CoV-2-S complex, shown in Figure 11B, the RMSF at the loop region was 5.6Å with a few ligand contacts (green-coloured vertical bars). This justifies the interactions seen in molecular docking. Further, in the RMSF plot for the RBD domain of the GA/SARS-CoV-2-S complex, shown in Figure 11C, the RMSF at loop region was 6.3Å with a high number of ligand contacts (green-coloured vertical bars), justifying the interactions seen in molecular docking. Though the ligand contacts are seen in interactions, the simulation time coverage determines their stability.\n\nProtein-ligand contacts. Protein-ligand interactions can be traced throughout the simulation and can be categorised into four types: hydrogen bonds, hydrophobic, ionic, and water bridges, as summarised in Figure 12A–12C. The stacked bars in the plots are normalised over the course of the trajectory and help us to understand the retention of contact throughout the simulation time. The contacts with a value of more than 0.7 are expected to be retained for over 70% of the total simulation time.\n\nA) streptomycin/SARS-CoV-2-S complex; B) ciprofloxacin SARS-CoV-2-S complex; C) glycyrrhizic acid/SARS-CoV-2-S complex. Desmond version 4.242. This analysis could also have been performed using open source software such as GROMACS43.\n\nIn the protein-ligand contact plot for the streptomycin/SARS-CoV-2-S complex, shown in Figure 12A, residues Glu493 and Lys544 showed maximum interactions fractions, i.e. 0.20 facilitated by hydrogen bonds and water bridges. This suggests that the specific interaction is maintained for 20% of the simulation time, and such short interactions are not promising. Hence, streptomycin cannot be a potential inhibitor of SARS-CoV-2-S to offer anti-COVID-19 activity.\n\nA) streptomycin/SARS-CoV-2-S complex; B) ciprofloxacin SARS-CoV-2-S complex; C) glycyrrhizic acid/SARS-CoV-2-S complex. Desmond version 4.242. This analysis could also have been performed using open source software such as GROMACS43.\n\nIn the protein-ligand contact plot for the ciprofloxacin/SARS-CoV-2-S complex shown in Figure 12B, residues Phe465, Tyr482, Tyr498, and Phe499 were seen to have the interactions fractions 0.75, 0.6, 0.35 and 0.39 respectively facilitated by hydrophobic, hydrogen bonds and water bridges. This suggests that for 70%, 60%, 35% and 39% of the simulation time, the specific interaction is maintained by respective residues and such interactions are considered good. Hence, ciprofloxacin may be a potential inhibitor of SARS-CoV-2-S and may offer anti-COVID19 activity.\n\nIn the protein-ligand contact plot for the GA/SARS-CoV-2-S complex shown in Figure 12C, residues Val492, Glu493, Asn496, Cys497, and Phe499 had the interactions fractions 0.78, 1.12, 0.80, 0.60 and 0.80, respectively, facilitated by hydrophobic, hydrogen bonds and water bridges. This suggests that for 78%,100%, 80%, 60% and 80% of the simulation time, the specific interaction is maintained by respective residues and such interactions are excellent and promising. Hence, GA can be a potential inhibitor of SARS-CoV-2-S and can offer anti-COVID19 activity.\n\n\nConclusion\n\nThrough our topological analysis, we have determined the degree of distribution for viral proteins, and we show that, due to its low degree of distribution, ACE2 is likely to be targeted by viruses like SARS-CoV. Hence, the interaction between the viral protein SARS-CoV-2-S and the host ACE2 receptor is a potential drug target for the repurposing of known drugs. Further, sequence alignment and domain analysis suggest that the RBD is the ligand-binding site. Molecular docking studies have suggested streptomycin, ciprofloxacin, and GA as possible leads to inhibit SARS-CoV-2-S. Molecular dynamic simulation analysis has indicated that GA is a promising small molecule that could be repurposed as a potential inhibitor of SARS-CoV-2-S to offer anti-COVID19 activity.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.", "appendix": "References\n\nZhou P, Yang XL, Wang XG, et al.: A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020; 579(7798): 270–273. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGorbalenya AE, Baker SC, Baric RS, et al.: Severe acute respiratory syndrome-related coronavirus: The species and its viruses - a statement of the Coronavirus Study Group. Nat Microbiol. 2020. Publisher Full Text\n\nWorld Health Organization: Clinical management of severe acute respiratory infection when novel coronavirus (CoV) infection is suspected. Reference Source\n\nCDC. Reference Source\n\nFDA. 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PubMed Abstract \| Publisher Full Text\n\nIlaria U, Elena A, Francesca T, et al.: Possible Antiviral Effect of Ciprofloxacin Treatment on Polyomavirus BK Replication and Analysis of Non-Coding Control Region Sequences. Virol J. 2013; 10: 274. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPompei P, Pani A, Flore O, et al.: Antiviral activity of glycyrrhizic acid. Experientia. 1980; 36(3): 304. PubMed Abstract \| Publisher Full Text\n\nSanner MF: Python: A Programming Language for Software Integration and Development. J Mol Graph Model. 1999; 17(1): 57–61. PubMed Abstract" }	[ { "id": "66947", "date": "14 Jul 2020", "name": "S.M. Vidya", "expertise": [ "Reviewer Expertise Phytochemical screening", "Pharmacology", "Molecular docking" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the present study authors have made an effort to repurpose the known drugs against SARS-CoV-2. Although the study is well organized and presented, a few minor changes are suggested for betterment.\n\nThe name of the molecules should begin with Uppercase.\n\nIn an abstract, authors have mentioned about the anti-viral activity of streptomycin, ciprofloxacin, and glycyrrhizic acid. But, it requires the literature support. Authors can cite the references for the anti-viral/ ant-microbial activity in Methods section.\n\nIn Results and Discussions, authors could claim the better affinity of glycyrrhizic acid towards SARS-CoV-2-S in comparison with other two molecules. Change suggested: Additionally, the ketone group of GA formed a hydrogen bond, with the backbone atoms of Phe499 receiving the electrons and possess the better affinity towards SARS-CoV-2-S when compared to Streptomycin and Ciprofloxicine.\n\nIn conclusion section, authors have mentioned that, ACE2 is likely to be targeted by viruses like SARS-CoV. Since, authors have tabulated the methods and references for ACE2 and SARS-CoV spike protein interaction in table 1, it is better to rewrite as \"ACE2 is known to be targeted by viruses like SARS-CoV\".\n\nIs the work 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": "67868", "date": "27 Jul 2020", "name": "Raghavendra HL", "expertise": [ "Reviewer Expertise Gestational Diabetes Mellitus", "Omics", "Drug Discovery", "Phytopharmacology and Bioinformatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript entitled “In silico screening of known small molecules to bind ACE2 specific RBD on Spike glycoprotein of SARS-CoV-2 for repurposing against COVID-19” addresses the repurposability of known drugs against COVID-19. Spike glycoprotein is the most popular drug target for SARS-CoV-2 having a significant role in host entry. With this basic understanding, the manuscript is reviewed and the observations are listed below.\nAbstract: Conclusion:\n\nThe statement “Hence, the molecules screened for inhibitory properties against SARS-CoV-2-S can be clinically used to treat COVID-19 since the safety profile is already known.” can be rewritten as “The GA of plant origin shown superior interaction with SARS-CoV-2-S compared to rest other molecules, hence, GA can be clinically investigated to confirm its efficacy to treat COVID-19.”\nMethods: Homology modelling of SARS-CoV-2-S protein\nThe crystal structure of SARS-CoV-S and ACE2 complex (PDB ID: 6ACD) is used as a template for homology modelling. It is expected to discuss the identity, query coverage between the query and template. Discuss the criteria for template selection.\nIn the figure legend for Figures 8c, 9c, 10c, 11c, and 12c glycyrrhizic acid can be replaced by GA.\nThe manuscript can be accepted for indexing with above-mentioned 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": [] } ]	1	https://f1000research.com/articles/9-663
https://f1000research.com/articles/9-157/v1	28 Feb 20	{ "type": "Software Tool Article", "title": "Visualize omics data on networks with Omics Visualizer, a Cytoscape App", "authors": [ "Marc Legeay", "Nadezhda T. Doncheva", "John H. Morris", "Lars Juhl Jensen", "Nadezhda T. Doncheva", "John H. Morris" ], "abstract": "Cytoscape is an open-source software used to analyze and visualize biological networks. In addition to being able to import networks from a variety of sources, Cytoscape allows users to import tabular node data and visualize it onto networks. Unfortunately, such data tables can only contain one row of data per node, whereas omics data often have multiple rows for the same gene or protein, representing different post-translational modification sites, peptides, splice isoforms, or conditions. Here, we present a new app, Omics Visualizer, that allows users to import data tables with several rows referring to the same node, connect them to one or more networks, and visualize the connected data onto networks. Omics Visualizer uses the Cytoscape enhancedGraphics app to show the data either in the nodes (pie visualization) or around the nodes (donut visualization), where the colors of the slices represent the imported values. If the user does not provide a network, the app can retrieve one from the STRING database using the Cytoscape stringApp. The Omics Visualizer app is freely available at https://apps.cytoscape.org/apps/omicsvisualizer.", "keywords": [ "Cytoscape", "app", "network visualization", "omics data", "network biology" ], "content": "Introduction\n\nCellular functions are mediated by complex networks of interactions between genes, proteins, and other molecular entities. Omics technologies are commonly used to measure the detailed regulation of these networks by quantifying changes of individual post-translational modification (PTM) sites, peptides, or splice isoforms across different experimental conditions. However, it is not easy to visualize such data sets, which have multiple values per gene or protein, onto the networks using existing network visualization tools such as Cytoscape1 or Gephi2.\n\nTo address this, we present the new Omics Visualizer app for Cytoscape3. The app allows users to import data tables with several rows referring to the same node and to visualize such data on networks; while designed with omics data in mind, the app is data agnostic. These values can be shown directly on the nodes of the networks as pie or donut visualizations, in which the color of each slice represents a different value for the same node. The Omics Visualizer app was implemented using the API that Cytoscape makes available to developers, and the data visualization builds upon the enhancedGraphics app4. The app furthermore integrates with the stringApp5 to facilitate easy visualization of data onto networks from the STRING database6 and supports Cytoscape Automation to facilitate integration with other tools7.\n\n\nMethods\n\nThe typical Omics Visualizer3 workflow consists of four steps: importing data as a table, optionally filtering the data, connecting it to one or more networks, and finally visualizing the connected data onto the networks.\n\nThe Omics Visualizer table import mimics the Cytoscape default import process. The app can handle text files (e.g. comma- or tab-delimited values) as well as spreadsheet files from e.g. Microsoft Excel. The app auto-detects the data type of each column, and the user can subsequently select which columns to import and modify the auto-detected data type for each column if needed. The import creates two private unassigned tables in Cytoscape: one that contains the data imported from the file, and another that stores all associated Omics Visualizer properties. These tables are private, which means that the user cannot interact with them directly in the Cytoscape UI. Instead, the data can be viewed (but not edited) through the Omics Visualizer panel located next to the node, edge, and network table panels. The tables are also accessible via the command interface and the Cytoscape API.\n\nIf the data was not already filtered before importing it into Cytoscape, Omics Visualizer enables users to do so afterwards. Omics Visualizer can filter the rows based on selected columns with string, numeric and Boolean values and offers several operators depending on the data type: EQUALS, NOT_EQUALS, NULL and NOT_NULL for all types; CONTAINS, NOT_CONTAINS and MATCHES for string values; LOWER, LOWER_EQUALS, GREATER and GREATER_EQUALS for numeric values. Similar to the Cytoscape selection filters, Omics Visualizer interface allows the user to create Boolean formulas with nested criteria. Once the filter is applied, rows that do not satisfy the filter are hidden in the panel, and the active filter is stored in the properties table to allow it to be changed later.\n\nTo visualize the data table onto a network, the Omics Visualizer table must first be connected to the network by specifying matching key columns in the node and Omics Visualizer tables. This connection information is stored in the network table, which is used to recreate the link when a session file is loaded. An Omics Visualizer table can be connected to several networks, but a network can only be connected to one Omics Visualizer table. A node table key can match the key in several Omics Visualizer table rows, in which case the node is connected to all of them. The number of connected rows for each node is stored in the node table; note that this number does not reflect any filtering.\n\nAlternatively, Omics Visualizer can retrieve and automatically connect a network from the STRING database6 using the Cytoscape stringApp5. The user can select any column with gene/protein identifiers, which will be used to first query STRING to retrieve a network and subsequently as the key for connecting the network to the Omics Visualizer table.\n\nOnce connected, the user can show the data from the Omics Visualizer table on networks using either a pie or a donut visualization. The data values are mapped to colors using either a discrete mapping, where the user chooses the colors for each value, or a continuous mapping, where the user defines a color gradient. For the continuous mapping, the user specifies three values and corresponding colors, namely the minimum, middle, and maximum. Every value lower than the minimum or greater than the maximum value will be shown using the minimum and maximum color, respectively. Predefined color mappings can be used with the help of Cytoscape palettes such as ColorBrewer8 and Viridis (originally from Matplotlib9). The charts are drawn thanks to the enhancedGraphics app4. This app draws charts based on a description string, which is specified as a Cytoscape Custom Chart node style.\n\nWhen the user creates a visualization, Omics Visualizer creates a column in the network table and several columns in the node table, and then it activates a Custom Chart node style: Omics Visualizer uses Custom Chart 7 to visualize pies and Custom Chart 8 to visualize donuts. The network table column is used to store the visualization properties. One node table column is created to store the enhancedGraphics string. Several node table columns are created to store the values of the visualizations: one column for pie visualization, and one column per ring for donut visualization. The values from the Omics Visualizer table rows are formatted into lists to fit the enhancedGraphics requirements. The enhancedGraphics continuous mapping always ranges from minimum to zero, and zero to maximum. Omics Visualizer thus first centers the values around the middle value and adjusts the minimum and maximum accordingly. The values are only modified in the node table columns, not in the actual Omics Visualizer table.\n\nOmics Visualizer can automatically generate legends from the visualizations in the form of Cytoscape annotations, which can be exported as part of the images if the user exports the network. To allow users to easily modify the legend, each element of the legend is a separate annotation. These are grouped so that the user can move or delete the legend in one click. The name of the annotation group is used to differentiate the legend annotations from other annotations and should thus not be changed. When the legend is created, Omics Visualizer creates a network table column to store it.\n\nAll the columns created by Omics Visualizer in the node or network tables are in the namespace \"Omics Visualizer\", enabling the user to easily identify and hide them if desired.\n\nOmics Visualizer is built from Java 1.8 using Cytoscape API 3.7, meaning the minimum required version of Cytoscape is 3.7.0.\n\nCytoscape can be used with R or Python through to Cytoscape Automation7. Omics Visualizer implements commands in the specific ’ov’ namespace, allowing Omics Visualizer to be used with the REST API. A full documentation of the commands is available at https://github.com/marclegeay/omics-visualizer/blob/master/automation_documentation.md. With the commands, the users can import a table, filter it, connect it with an existing network, retrieve a STRING network, create visualizations, and generate legends.\n\n\nUse cases\n\nWe will illustrate how to use Omics Visualizer3 by visualizing site-specific proteomics data from a phosphoproteomics study of ovarian cancer by Francavilla et al.10. This study compares the phosphoproteome of primary cells derived from epithelial ovarian cancer (EOC) and two healthy tissues, namely ovarian surface epithelium (OSE) and distal fallopian tube epithelium (FTE). The goal of the study was to uncover cancer-specific changes in expression, phosphorylation state, and kinase signatures by comparing cancer and two healthy tissues (EOC vs. OSE, and EOC vs. FTE) for each site. The sites were then clustered into three clusters: A) sites abundant in healthy (OSE and FTE) but not cancerous tissues (EOC); B) sites abundant in FTE and EOC, but not OSE; and C) sites abundant only in cancerous tissue (EOC).\n\nIn this example, we want to visualize this site-specific data set on a network in which each node is a protein. We start from Table S3 from Francavilla et al., where each line represents a phosphorylation site in a protein, as opposed to a protein. We will first load the table file into Cytoscape thanks to Omics Visualizer and then create the network from the data. We will finally apply some style to the network to visualize the data.\n\nTable S3 only contains the expression in the different samples, but not the individual comparisons between EOC and healthy tissues, so we modified it to add the comparison columns ’EOC vs OSE’ and ’EOC vs FTE’. The modified file is avialable as Underlying data11.\n\nTo load the data, we will use the specific import feature from Omics Visualizer, that can be accessed from the ’File → Import → Omics Visualizer table from File...’ menu, or the ’Apps → Omics Visualizer → Import table from file’ menu. This opens a custom dialog, which is very similar to the standard Cytoscape import table dialog. The user can name the table (or a default name will be given) and select the columns and their type to import. By default, all columns are imported and their type is inferred according to the first hundred lines of the file.\n\nHere, we specifically want to import the ’UniProt’ column to retrieve a STRING network afterwards, the ’AA Position’ column to label the sites, the comparison columns ’EOC vs OSE’ and ’EOC vs FTE’ with log-ratios to be visualized, the ’Adj p-value’ column to filter the table by significant sites, and the cluster assignment column ’Cluster’ to visualize it. The file can be loaded with the following command:\n\n\n\nOnce the table is imported, the Omics Visualizer panel appears (Figure 1, left table). The top part of the panel has a row of icons giving access to the different features, displays the number of rows of the table, and enables access to the different Omics Visualizer imported tables. The second part of the panel is the table itself.\n\nBoth tables can be linked together using the key column \"UniProt\" from the Omics Visualizer table with the key column \"query term\" from the node table. The color boxes identify the key values used to link the two tables, the color lines emphasize the link between the rows of the two tables.\n\nIt is possible to filter the table with the GUI or with the automation command. The filter GUI can be accessed via the filter icon from the ’Omics Visualizer Tables’ tab, or with the ’Apps → Omics Visualizer → Filter table’ menu. We filter the table so that we select only sites that have an adjusted p-value lower or equal to 0.01. This filter can be applied to the current table with the following automation command:\n\n\n\nAfter the filter has been applied, the filter icon changes colors and the number of rows before and after filtering is displayed (Figure 1, left table).\n\nIn this use case, we will use a STRING network corresponding to our data. With the help of the stringApp, we perform a ’protein query’ with the list of the UniProt identifiers of proteins from our table that have at least one phosphorylation site with an adjusted p-value lower or equal to 0.01.\n\nThe current version of the stringApp retrieves a STRING v11 network with default confidence of 0.4 that consists of 237 nodes and 1020 edges. To reduce the size of the network, we cluster it using the Markov clustering from the clusterMaker2 app12. We used the inflation value of 2.5 and the stringdb score as array sources. For illustrations purposes, we here show only the second biggest cluster, which contains 40 nodes and 107 edges.\n\nOnce the STRING network is imported in Cytoscape by the stringApp, we connect the network with the table. The link icon or the ’Apps → Omics Visualizer → Manage table connections’ menu give access to the connect dialog. The dialog shows already connected networks and gives the possibility to modify or delete them. You can also create a new connection by selecting the network to connect to, then the two key columns from the network and from the table. In our case, the network was retrieved from the UniProt identifiers stored in the ’UniProt’ column of our table. The stringApp stores the query into the column ’query term’, so we use it as ’key column from Network’ and we use ’UniProt’ as the ’key column from Table’. Figure 1 illustrates the mapping between the two tables. The \"P20700\" (blue box) from the node table is linked to two rows of the Omics Visualizer table which have the same value in the UniProt column.\n\nThe connection between the current network and the current table can be performed with the following automation command:\n\n\n\nIt is possible to do the previous step more quickly by automatically retrieving a STRING network from the Omics Visualizer table. We can retrieve a STRING network with the icon or the ’Apps → Omics Visualizer → Retrieve STRING network’ menu. We have to select the species, the column that contains the identifiers to query, the confidence cutoff and, if filtering was applied as in our case, whether to retrieve the identifiers only from filtered rows. By default the species is human, the cutoff is 0.40, and the query column is identified by a case-insensitive search for \"uniprot\" among the column names.\n\nOnce the STRING network is retrieved thanks to stringApp automation, the network is automatically connected to the Omics Visualizer table with the ’query term’ column from the node table with the query column selected by the user. The network can also be retrieved from the current Omics Visualizer table with the following automation command:\n\n\n\nThe visualization is configured thanks to a specific dialog, that can be reached with the donut icon or the ’Apps → Omics Visualizer → Create donut visualization’ menu.\n\nHere, we want to visualize the two disease vs. healthy comparisons for each site. We select as Values the two comparison columns (EOC vs FTE and EOC vs OSE) that contain the numerical values we want to visualize. Because the values are log-ratios, we apply a continuous mapping and use a color scale. To be able to identify the individual phosphorylation sites in the network, we label the charts with the position of the amino acid (AA position column) of the site in the sequence of the protein. We customize the chart so that the first slice starts at a 3 o’clock angle and the label font size is changed to 15. The next dialog enables the user to configure the color scale. By default, a diverging palette is selected, and the color scales from the minimum to the maximum value and is centered on zero. The minimum and maximum bound values are directly computed from the values of the table: the maximum bound is the maximum of the absolute value of the minimum and maximum data values; the minimum bound is the opposite of the maximum bound. The user can change both the values and the colors associated. In our case, we will change the range and set it from -8 to 8. If a table value is lower than the minimum value of the scale or larger than the maximum, the value will be associated with the color associated with the minimum or maximum value, respectively. The visualization can be modified or deleted by accessing the dialog again. The resulting network can be seen in Figure 2. We have two donuts around each node representing the two comparisons. One slice of a donut is one value from the Omics Visualizer table associated with the node: in our case, it is the log-ratio of a specific site. It is possible to flip the visualization, and have as many donuts as sites, with two slices for each comparison.\n\nLog-ratios of protein abundance between cancer and healthy tissues were mapped around the nodes using a blue–white–red gradient. Each slice of the rings represents a significantly regulated phosphorylation site. The inner ring is the comparison between epithelial ovarian cancer and distal fallopian tube epithelium (EOC vs FTE), the outer ring is the comparison between epithelial ovarian cancer and ovarian surface epithelium (EOC vs OSE). This subnetwork is the second biggest cluster obtained using Markov clustering on the STRING network of all proteins from the study that have at least one phosphorylation site with an adjusted p-value lower or equal to 0.01.\n\nThe visualization of the current table can be obtained with the following automation command:\n\n\n\nIf we are not interested in the individual log-ratios from the comparisons, we can instead summarize the results as a pie visualization of the expression clusters.\n\nThe inner visualization can be configured thanks to the pie icon or the ’Apps → Omics Visualizer → Create pie visualization’ menu. The inner visualization dialog looks like the outer visualization dialog, and the user must first select the column that contains the values to draw. The difference here is that the user can only select one column, because the different slices of the pie come from the different values associated with the same node. In our case, we want to visualize the \"Cluster\" column and apply a discrete mapping. As for the outer visualization, we will label the chart slices with the amino acid position (AA position). We also customize the chart to have the first slice starting at a 3 o’clock angle and change the label font size to 15. The next dialog enables us to configure the color mapping. By default, Omics Visualizer selects a qualitative palette. Here the user may change the color associated with each value detected in the table. The default colors are too similar to each other and we changed them by clicking on the color square. The inner visualization can also be modified or deleted by accessing the dialog again.\n\nThe resulting network is shown in Figure 3 and can be obtained with the following command:\n\n\n\nEach slice of a pie corresponds to a significantly regulated phosphorylation site of the protein and the color of the slice represents the cluster to which the site belongs. Sites abundant in healthy (OSE and FTE), but not cancerous (EOC) tissues are in cluster A; sites abundant in FTE and EOC, but not OSE are in cluster B; and sites abundant only in EOC are in cluster C.\n\nThe user can generate a legend by clicking the map icon and choosing which legends to generate (inner and/or outer visualizations), the title, the font, and the position of the legend. Omics Visualizer will then create Cytoscape annotations corresponding to the current visualizations, displaying names of the columns above the color legends. In the case of the outer visualization, a list of column names shows the order of the columns. Because legends are annotations, they can be moved, edited, and exported with the network as an image.\n\nThe legends in Figure 2 and Figure 3 were automatically generated with the following command:\n\n\n\n\nConclusions\n\nOmics Visualizer3 is a Cytoscape app that improves how omics data can be visualized on molecular networks. A key feature of Omics Visualizer is that, unlike the standard Cytoscape node table, it enables the user to import files with several rows of data related to the same node, with each row representing a different site, isoform, or experimental condition. This data can subsequently be visualized in or around the nodes as charts with a separate slice for each row that is connected to the node. These slices can be colored to show both continuous and discrete values, and Omics Visualizer can automatically produce a color legend to help in preparation of publication-ready figures. All the actions of Omics Visualizer can be done using the Cytoscape GUI or the commands, making it easy to use Omics Visualizer also in R or Python via the Cytoscape REST API.\n\n\nData availability\n\nZenodo: Modified Table S3 - Francavilla et al. 2017. https://doi.org/10.5281/zenodo.362922011.\n\nData is available under the terms of the Creative Commons Attribution 4.0 International.\n\nThe software, source code, and tutorial are available at the Cytoscape App Store: http://apps.cytoscape.org/apps/omicsvisualizer.\n\nArchived source code at the time of publication: https://doi.org/10.5281/zenodo.36315843.\n\nLicense: BSD 2-Clause \"Simplified\" License.", "appendix": "Author contributions\n\n\n\nJHM and LJJ designed the app. ML is the main developer of the app, helped by NTD. NTD, JHM and LJJ contributed to the manuscript. ML wrote the manuscript.\n\n\nAcknowledgements\n\nThe authors thank Katerina Nastou and Giulia Corsi for beta-testing the app.\n\n\nReferences\n\nShannon P, Markiel A, Ozier O, et al.: Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003; 13(11): 2498–2504. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBastian M, Heymann S, Jacomy M: Gephi: An open source software for exploring and manipulating networks. International AAAI Conference on Weblogs and Social Media. 2009. Publisher Full Text\n\nLegeay M: Omics Visualizer. 2020. http://www.doi.org/10.5281/zenodo.3631584\n\nMorris JH, Kuchinsky A, Ferrin TE, et al.: enhancedGraphics: a Cytoscape app for enhanced node graphics [version 1; peer review: 2 approved]. F1000Res. 2014; 3: 147. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDoncheva NT, Morris JH, Gorodkin J, et al.: Cytoscape StringApp: Network Analysis and Visualization of Proteomics Data. J Proteome Res. 2019; 18(2): 623–632. PubMed Abstract \| Publisher Full Text \| Free Full Text\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–D613. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOtasek D, Morris JH, Bouças J, et al.: Cytoscape Automation: empowering workflow-based network analysis. Genome Biol. 2019; 20(1): 185. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHarrower M, Brewer CA: ColorBrewer.org: An Online Tool for Selecting Colour Schemes for Maps. Cartogr J. 2003; 40(1): 27–37. Publisher Full Text\n\nHunter JD: Matplotlib: A 2D Graphics Environment. Comput Sci Eng. 2007; 9(3): 90–95. Reference Source\n\nFrancavilla C, Lupia M, Tsafou K, et al.: Phosphoproteomics of Primary Cells Reveals Druggable Kinase Signatures in Ovarian Cancer. Cell Rep. 2017; 18(13): 3242–3256. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLegeay M: Modified Table S3 - Francavilla et al. 2017. 2020. http://www.doi.org/10.5281/zenodo.3629220\n\nMorris JH, Apeltsin L, Newman AM, et al.: clusterMaker: a multi-algorithm clustering plugin for Cytoscape. BMC Bioinformatics. 2011; 12(1): 436. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "60743", "date": "10 Mar 2020", "name": "Magnus Palmblad", "expertise": [ "Reviewer Expertise Analytical chemistry", "molecular biology", "mass-spectrometry based proteomics", "bioinformatics." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTo evaluate the user experience, I first downloaded and used the Omics Visualizer, following the on-line tutorial. The interface is intuitive (like how Cytoscape itself works), exposing just the right amount of information and options to the user. It is also clear to the user what the app does.\n\nThe manuscript partially overlaps with but complements the on-line tutorial with background and some technical detail how the app integrates in Cytoscape. The Omics Visualizer fills a clear niche in the Cytoscape appverse, allowing tables to be imported surjectively (mapping multiple rows in the input to the same network node) as opposed to bijectively (with a one-to-one correspondence between the rows in the input table and nodes in the network). I suppose this could be achieved by manipulating tables (connecting row information in the data to columns in a node table) in R or Python, pushing the necessary data to Cytoscape through RCy3 or py2cytoscape (Omics Visualizer commands are also exposed via the Cytoscape REST API). But the Omics Visualizer is completely interactive and requires no scripting or formulae to be entered in the Cytoscape tables. The menus are clear and self-explanatory. The visualizer provides pie and donut visualizations for displaying multiple node attributes, such as post-translational modification sites and occupancy onto the same node. As would be expected from this team, Omics Visualizers also works well with the Cytoscape STRING app. I find this manuscript a very valuable companion to the app and on-line tutorial.\nThe manuscript is very clearly written, and the length appropriate for the subject matter. I only have two minor comments:\n\"3 o’clock angle\" should be \"3 o’clock position\" (in the \"Outer\" and \"Inner visualization\" paragraphs)?\n\nThe \"Filter table rows\" option referred to in the on-line tutorial is called \"Filter table\" in Cytoscape.\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": "5627", "date": "30 Jun 2020", "name": "Marc Legeay", "role": "Author Response", "response": "We would like to thank you for your comments and we have modified the manuscript to take them into account. In the meantime, we also released a new version of the app with the additional feature to import columns from the node table of a network loaded in Cytoscape. We thus further modified the manuscript to describe the new feature. We also added two new figures to describe the general Omics Visualizer workflow and to clarify the format needed for input tables." } ] }, { "id": "61035", "date": "15 Apr 2020", "name": "Ruth Isserlin", "expertise": [ "Reviewer Expertise bioinformatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAuthors Legeay et al. introduce a new Cytoscape app to enable easy import and visualization of multi data type -omics data onto to networks with added features to quickly import interaction data from String. A great new addition to the cytoscape app set.\n\nDetailed comments:\nIn the method section -\n\n“the data can be viewed (but not edited) through the Omics Visualizer panel located next to the node, edge, and network table panels.” - not sure what you mean by this. Is it an additional tab in the table panel or is it its own panel next to the table pane? After running the app I see that it is an additional tab. it might sound better as “the data can viewed (but not edited) through the Omics Visualizer panel in the table panel.” (Just for aesthetics I really like it if the app icon can be included in the tab name as well. Makes it easier to spot and click on.)\n“but a network can only be connected to one Omics Visualizer table.” - Does this mean that you can’t annotate a network with isoform data and phosphorylations at the same time? You can use both pie and donut visualizations at the same time so as long as the different data types are represented in the same table you can use multiple types. Maybe specify that an network can only be associated with one omics set but if you integrate multiple omics data into one table prior to loading into cytoscape you can integrate different data types. The paragraph starting with “To visualize the data table onto a network,” that outlines how the different tables are connected and the relationships of those connections would be much clearer with a figure outlining all the different connections described.\n\nIt would be helpful to have a description of the type of table the app is expecting and how the two different charts can be used. I.e. I loaded in a table with genes vs. Patients expecting to see all my patient data on the node but I think that it only maps one column for the pie chart and multiple columns for the donut so the expectation is that there will be duplicate genes in a given column for the pie.\nWhat is the maximum number of conditions/instances that can be mapped to an individual node and visualization type?\n“The enhancedGraphics continuous mapping always ranges from minimum to zero, and zero to maximum.” - are minimum values calculated based on the filtered set of -omics data or the entire dataset loaded?\n“Omics Visualizer can automatically generate legends from the visualizations in the form of Cytoscape annotations, which can be exported as part of the images if the user exports the network. “ - it was very difficult to find the legend. I had to come back to the text a few times to figure out that it was somewhere on the network canvas and compared to the network it was tiny so it was hard to find.\n\nUse cases:\n“To reduce the size of the network, we cluster it using the Markov clustering from the clusterMaker2 app” - in the clustermaker2 app Markov clustering is called MCL. Might be good to add MCL in brackets for users who don’t know they are equivalent.\n“For illustrations purposes, we here show only the second biggest cluster, which contains 40 nodes and 107 edges.” - it is not clear how you limit to just that cluster. Do you filter the network by __mclCluster or do you create a network of the clusters directly from the cluster maker2 app?\n“It is possible to flip the visualization, and have as many donuts as sites, with two slices for each comparison.” - how could this be done? Does it involve changing our choice of column to EOC and FTE and OSE separately?\nInner and outer visualization can be confusing. At first I thought you were referring to the inner and outer rings of the donut. Maybe changing or adding Pie and Donut to Outer visualization and Inner visualization headers will be helpful.\n“The difference here is that the user can only select one column, because the different slices of the pie come from the different values associated with the same node. “ - This is an important distinction that needs to be specified earlier. Whether it is in the introduction or in the methods but I think that the different type of data Omics is expecting and the data types best used for each visualization types needs to be expanded on sooner in the paper.\nIn the inner visualization section - “In our case, we want to visualize the \"Cluster\" column and apply a discrete mapping.” - it is unclear what the meaning for this “Cluster” column that was in the initial file loaded.\nIn the legend section - “Because legends are annotations, they can be moved, edited, and exported with the network as an image.” - how can the legends be edited or moved? I tried right clicking on the legend and selected Edit -> modify annotation but nothing happened. I figured out that you modify the annotation through the annotation panel but might be good to add how you do it here. (I couldn’t figure out how to move it though)\nMinor grammatical suggestions:\n“For illustrations purposes, we here show only the second biggest cluster, which contains 40 nodes and 107 edges.” - remove the “here” “For illustrations purposes, we show only the second biggest cluster, which contains 40 nodes and 107 edges.”\n“The visualization is configured thanks to a specific dialog” - not sure what this is saying.\n“The inner visualization can be configured thanks to the pie icon or the ’Apps → Omics Visualizer → Create pie visualization’ menu. “ - sounds better as “The inner visualization can be configured using the pie icon or the ’Apps → Omics Visualizer → Create pie visualization’ menu.”\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": "5628", "date": "30 Jun 2020", "name": "Marc Legeay", "role": "Author Response", "response": "We would like to thank you for your comments and suggestions. We have addressed them in the new version of the manuscript, which also describes a new feature added to the app during review, namely to import columns from the node table of a network loaded in Cytoscape. We also added two new figures to describe the general Omics Visualizer workflow and to clarify the format needed for input tables. We took into account all your comments regarding the phrasing. For other comments, we give our answers below (we labeled your comments with “Reviewer” and ours with “Response”). Reviewer: “but a network can only be connected to one Omics Visualizer table.” - Does this mean that you can’t annotate a network with isoform data and phosphorylations at the same time? You can use both pie and donut visualizations at the same time so as long as the different data types are represented in the same table you can use multiple types. Maybe specify that an network can only be associated with one omics set but if you integrate multiple omics data into one table prior to loading into cytoscape you can integrate different data types. Response: Right now, a network can only be connected to one Omics Visualizer table, so indeed if the user wants to have both visualizations, all the data has to be merged into one table (see new Figure 2C). We are currently working on a new version of the app that would enable a network to be connected to several tables. Reviewer: It would be helpful to have a description of the type of table the app is expecting and how the two different charts can be used. I.e. I loaded in a table with genes vs. Patients expecting to see all my patient data on the node but I think that it only maps one column for the pie chart and multiple columns for the donut so the expectation is that there will be duplicate genes in a given column for the pie. Response: We added a new Figure 2 giving three examples of tables that could be imported into Omics Visualizer. We hope it makes things easier to understand. Reviewer: What is the maximum number of conditions/instances that can be mapped to an individual node and visualization type? Response: There is no technical limit to the number, and it is thus up to the user to decide how many slices or rings to make. However, to create easily understandable visualizations, we would advise against having excessively many slices or rings. Reviewer: “The enhancedGraphics continuous mapping always ranges from minimum to zero, and zero to maximum.” - are minimum values calculated based on the filtered set of -omics data or the entire dataset loaded? Response: The user can put a minimum and maximum value, all values below the minimum will be colored as the minimum, and all values above the maximum will be colored as the maximum. Those values are, by default, the min and max of the filtered values. The color gradient from enhancedGraphics ranges from a negative value to zero, then zero to a positive value. Thus, Omics Visualizer has to change the table values to center the data. We modified the manuscript accordingly. Reviewer: “Omics Visualizer can automatically generate legends from the visualizations in the form of Cytoscape annotations, which can be exported as part of the images if the user exports the network. “ - it was very difficult to find the legend. I had to come back to the text a few times to figure out that it was somewhere on the network canvas and compared to the network it was tiny so it was hard to find. Response: By default, the font size of the legend is the same as the font size of the node labels, meaning that if you have a huge network, the legend with default font size can be hard to find. When the legend is generated, we change the view of the network to fit everything (including the legend). Reviewer: “For illustrations purposes, we here show only the second biggest cluster, which contains 40 nodes and 107 edges.” - it is not clear how you limit to just that cluster. Do you filter the network by __mclCluster or do you create a network of the clusters directly from the cluster maker2 app? Response: We enabled the option “Create new clustered network” when we ran clusterMaker and extracted the second largest cluster by selecting all nodes and edges belonging to it and creating a new network from the selection. Reviewer: “It is possible to flip the visualization, and have as many donuts as sites, with two slices for each comparison.” - how could this be done? Does it involve changing our choice of column to EOC and FTE and OSE separately? Response: No, you can keep the same columns, it is just a matter of whether each ring represents a condition (“ring as column”) or a site (“ring as row”). Reviewer: In the legend section - “Because legends are annotations, they can be moved, edited, and exported with the network as an image.” - how can the legends be edited or moved? I tried right clicking on the legend and selected Edit -> modify annotation but nothing happened. I figured out that you modify the annotation through the annotation panel but might be good to add how you do it here. (I couldn’t figure out how to move it though) Response: We added an explanation on how to move and change the text of the legend using Cytoscape’s Annotations control panel." } ] } ]	1	https://f1000research.com/articles/9-157
https://f1000research.com/articles/9-220/v1	31 Mar 20	{ "type": "Research Article", "title": "Antioxidant (gallic acid and quercetin) profile of Sumatran wild mangoes (Mangifera spp.): a potential source for antidegenerative medicine", "authors": [ "Fitmawati Fitmawati", "Esi Resida", "Sri Nur Kholifah", "Rodesia Mustika Roza", "Muhammad Almurdani", "Emrizal Emrizal", "Esi Resida", "Sri Nur Kholifah", "Rodesia Mustika Roza", "Muhammad Almurdani", "Emrizal Emrizal" ], "abstract": "Background: New findings on the potential of wild mangoes from the island of Sumatra as a source of antioxidant helps their conservation effort as it introduces their useful compounds to the public. This study aims to analyze the antioxidant profile and quantification of gallic acid and quercetin content from leaves and bark of Sumatran wild mangoes. Exploration and analysis of phytochemical constituents from 11 Sumatran wild mangoes was performed. Methods: Antioxidant activity of wild mangoes was analysed with 1,1- diphenyl-2-picryl hydroxyl (DPPH), and determination of quercetin and gallic acid content was performed by high performance liquid chromatography (HPLC) method. Total flavonoid and phenolic analysis was also performed. Curve fitting analysis used a linear regression approach. Results: The highest level of antioxidant activity, phenolic compound and flavonoid compound was found in the leaves and bark of Mangifera sp1. (MBS), the bark of M. foetida3 (var. batu) and leaves of M. torquenda, and the bark and leaves of M. sumatrana, respectively. The content of gallic acid in leaves ranged from 5.2270-35.4763 mg/g dry weight. Quercetin content of wild mangoes leaves ranged from 0.76 to 1.47 mg/g dry weight with the lowest value in M. foetida2 (var. manis) and the highest in M. laurina. Conclusion: The results obtained are expected to be useful in supporting the development of antidegenerative drugs from natural ingredients that have potential as immunomodulatory agents.", "keywords": [ "antioxidant", "gallic acid", "quercetin", "Sumatran", "wild mango" ], "content": "Introduction\n\nSumatran wild mangoes have the potential to be used as an herbal medicine and are found in the Sumatra rainforests. However, they are threatened with extinction due to rapid and massive deforestation, and due to this species lower economic market value and low consumption by humans, there is little interest in their cultivation1. This could lead to an increase in the number of endangered species in the Sumatran rainforest. In nature, these species are ecologically valuable as the primary food for primates and other wildlife.\n\nThere are eight wild mango species found in Sumatra: Mangifera quadrifida, M. torquenda, M. magnifica, M. grifithii, M. kemanga, M. sumatrana, Mangifera sp1 (MBS) and Mangifera sp2. (MH)2,3. The conservation of wild mango types will not be effective if the benefits of these plants are unknown, while their unique characteristics show that these plants have the potential to be therapeutic agents with high antioxidant ability4. Consequently, studying their ecological role and finding their potential biomedicine compounds is the best way to aid wild mango conservation.\n\nThese wild mangoes are acidic and have a strong turpentine aroma, rough fibers and strong odor, showing that these mangos are an antioxidant source as reported by Fitwawati et al.4. Antioxidants are vital to resolve some types of degenerative diseases, improving immunity and responding to external attacks, such as bacteria, viruses, fungi, and various disease-causing germs. They may even be used to manage chronic diseases, such as cancer. In the study by Taing et al.5, skin and flesh extracts of mangos (M. indica) had the remarkable action of inhibiting proliferation of breast and colon cancer cells6.\n\nPrevious studies show that mango skin (M. indica) possess flavonol O- and xanthone C-glycosides7, gallotannins and benzophenone derivatives8. Mango seeds contained bioactive components such as phenolics, carotenoids and ascorbic acid9, carbohydrates (58–80%), proteins (6–13%), essential amino acids and lipids (6–16%)10. The polyphenol component of mango extract is phenolic acid (gallic acid) and flavonoids (quercetin)11. These compounds are found in the edible fruits and have been shown to have great potential for protecting the body from oxidative stress-associated damages12,13.\n\nGallic acid (3,4,5-trihydroxybenzoid acid) is a major polyphenolic compound present in mangoes14. Phenolic compounds are vital in the immune system response to chronic degenerative diseases, such as anti-aging, anti-inflammatory, antidiabetic and antiproliferative activities, and have a cardioprotective effect, including reduction the side effects of chemotherapeutics15. Quercetin (3,3’4’,5,7-pentahydroxyflavone) is one of the most abundant flavonoid-derived food compounds and is present in various mangos (e.g. M. indica)16. Flavonoid structure contains a double bond in the C ring and a 4-oxo group, which enhances its potent antioxidant activity17. Flavonoids have been scientifically proven to be anti-allergic, anti-inflammatory, anti-cancer18, antimicrobial19 and antiviral20. Quercetin generally exists in edible plants and is mostly used in the production of traditional medicine to relieve some type of diseases21–23.\n\nPrevious research related to the antioxidant potential of wild mango species from Sumatra had been carried out qualitatively by Fitmawati et al.4. Yet, this research topic needs the support of quantitative data and detailed metabolite content in order to clarify the antioxidant compound of wild mangos that have high potential to be a source of antidegenerative drugs. To the best of our knowledge, quantitative analysis of the antioxidants in wild mangos has not yet been reported. Therefore, this study aimed to analyze the antioxidant compound (gallic acid and quercetin) of wild mangos from Sumatra, Indonesia in order to preserve its existence in nature. The results obtained are expected to be advantageous in the effort of discovering types that contain highly phenolic and flavonoid compounds, which will support antidegenerative drug development for public health. The finding of medicinal compounds in wild mangos may make stakeholders pay attention to cultivation and restoration, so their population in nature will be conserved.\n\n\nMethods\n\nEquipment. Preparative-plate glass for thin layer chromatography, Whatman filter-paper, vacuum rotary evaporator, ultraviolet lamps 254 and 366, UV-Visible spectrophotometer (GENESYS 10S UV-VIS), High Performance Liquid Chromatography (HPLC; Shimadzu LC 20AD), cuvette, micropipette. Materials: stem bark and leaves of wild mango from Sumatra, distilled water (Batraco), ethanol (Batraco; as solvent), silica gel GF254 (Batraco; for isolation), and ethyl-acetate (Batraco; as solvent).\n\nThe wild mango samples were collected from several provinces in Sumatra Island, such as Riau, Jambi and South Sumatera, as depicted in Figure 1. Wild mango bark was dried in an oven at 40°C, while the leaves were freeze-dried. Dried bark and leaves were ground by a blender and ~100 g of the resulting powder was macerated with 200 ml ethanol until it was submerged, and was then soaked for a day. All macerate was collected and evaporated with a rotary vacuum evaporator at 50°C to obtain a solid-liquid extract.\n\nAntioxidant activity analysis was assessed with the DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) method24. Briefly, approximately 2 mg of the dried sample was dissolved in 2 mL of methanol until the concentration reached 1000 mg/mL. Then, in a plate consisting of rows A-H (each row consisted of 12 wells), 100 mL of sample was added to each well in row A. Then, a total of 50 mL methanol was added to each well for rows B to F. 50 mL was taken from row A and then added to the row B; the same volume taken from row B and added to row C; the same procedure was done to row F. 50 mL was taken from row F and discarded. The following concentrations were obtained through this process: 1000 (row A), 500 (B), 250 (C), 125 (D), 62.5 (E), and 31.25 (F) g/mL. Row G to H were filled with 50 mL of methanol, only wells 1-6 of row H were filled. Furthermore, for DPPH test, additional methanol of 80 mL (concentration of 80 mg/mL) is added to the rows A to G, and then incubated for 30 minutes. Radical bounding was measured by the decrease in DPPH absorbance using a microplate reader at a wavelength of 517 nm. A positive control was used as a comparison (ascorbic acid, 50 ug/mL).\n\nThe antioxidant activity is presented as inhibition percentage of IC50, which was calculated by the formula below25:\n\n\n\nwhere A0 is the absorbance without sample and Ai is the absorbance of the tested sample.\n\nAnalysis of total flavonoid content was carried out using the method of Xu and Chang26. Quercetin solution was firstly dissolved into five concentrations (20, 40, 60, 80 and 100µg/ml) for a standard curve. Subsequently, ~100 μl of quercetin (blank) sample was mixed together with 50 NaNO2 5% and 50 μl AlCl3.6H2O (aluminum chloride hexahydrate) 10% and 50 μl NaOH 1 M were added to a 96 well clear polystyrene microplate. The mixture was incubated in a dark place at room temperature for 30 minutes. After that, the mixture was assessed using a microplate reader at a wavelength of 510 nm. Total flavonoid content is determined by following equation:\n\n\n\nwhere b and Dp are regression coefficient and dilution factor, respectively, V is extracted volume of the sample, and mt is extracted mass. Note that the mango sample is in dried form (100g used), so that mtotal refers to total mass of extracted sample and mdry indicates sample dry mass.\n\nAnalysis of total phenolic contents from dried samples of 100 gr was performed using the method of Folin-Ciocalteu from Musa et al.27. Gallic acid solution was initially dissolved into five concentrations (20, 40, 60, 80, and 100µg/ml) for the standard curve. About 100 μl of hydrolysis and non-hydrolysis of gallic acid as reference sample was then mixed with 50 μl of Folin-Ciocalteu reagent 0.25 in a 96 well clear polystyrene microplate. Next, 50 μl Na2CO3 7,5% was added to each well. The mixture was subsequently incubated in a dark place at room temperature for 30 minutes and was read using a microplate reader at a wavelength of 765 nm. Total content of phenolic is calculated by the same equation used for determining flavonoid content as in Equation 2.\n\nMango extract was dissolved in ethanol (100 g/100 mL). Quercetin and gallic acid standard compounds were each prepared in several concentrations of 100, 50, 25, and 12.5 ug/ml. Each standard compound was mixed with 20 μL and eluted for 20 minutes through elution gradient method (with water/methanol = 0-100 and UV detector with wavelengths of 360 and 370 nm) to obtain a standard chromatogram. The sample was then filtered using a 0.45 μm filter (13mm PTFE) and analyzed according to the HPLC method of quercetin and gallic acid. The level of quercetin and gallic acid contained in each sample was calculated from regression plot of Y= a lnX+b. This plot is described the ratio of the sample concentration to the standard chromatogram of the quercetin and gallic acid standard.\n\nThe content of gallic acid and quercetin in the sample is indicated by the change of the solution color in DPPH test which indicated antioxidant activity28. This change was read by a microplate (Berthold, Germany) using the software of MikroWin 2000 version 4.3x. Furthermore, a linear regression curve is performed to obtained quantification of gallic acid and quercetin content from each sample.\n\n\nResults\n\nAntioxidant values in this study were determined using the DPPH method. This method is a simple, easy, fast and sensitive method. Only a small amount of natural material is used to evaluate antioxidant activity, so it is widely used to test the ability of compounds that act as electron donors28. The principle of this measurement is the presence of stable free radicals, namely DPPH mixed with antioxidant compounds that have the ability to donate hydrogen so that free radicals are neutralized29. Measurement of antioxidant activity using UV-vis spectrophotometry is used so that the value of free radical will be known, expressed by the value of IC50 (inhibitory concentration). Table 1 contains the antioxidant activity of eight wild mango species.\n\nAntioxidant compounds will generally react to DPPH radicals through mechanism of donation of hydrogen atoms which causes the discoloration from purple to yellow28. In this study, the strongest levels of antioxidant activity in wild mangos was found in Mangifera sp1. (MBS) leaves and bark extract with IC50 value of 0.88 ppm and 33.24 ppm, respectively. Antioxidant compounds at a moderate level were discovered in the extract of Mangifera foetida1 (var. limus) leaves (IC50 value of 196.24 ppm) and Mangifera foetida2 (var. manis) bark (IC50 value of 117.62 ppm).\n\nThe phenolic and flavonoid compounds were quantified in the mangos using the method of Folin-Ciocalteu from Musa et al.27 and the method of Xu and Chang26, respectively. Total phenolic content was represented in milligrams (mg) of gallic acid, which was equivalent to per gram of the dry weight of mango leaves (mg GAE/g) (Table 2). Total flavonoid content was represented in milligrams (mg) of quercetin, which was equivalent to per gram of the dry weight of mango leaves dry weight (mg of QE/g).\n\nSpectrum chromatogram of standard solutions and chromatograms of each species at wavelengths of 360 and 370 nm were used. Specific quantitative tests of gallic acid and quercetin on the leaves of wild mangos from Sumatra using HPLC showed that the plant leaves were positive for both bioactive compounds. The plot of standard calibration fo quercetin and gallic acid is shown in Figure 2 and the results are summarized in Table 3.\n\n\nDiscussion\n\nAntioxidants are a group of chemical compounds that have the function of suppressing cell damage, caused by free radicals, by giving electrons speedily and transforming the free radicals into stable forms, in order to prevent oxidative damage that cause ailments30. The presence of antioxidants in plants is primarily as a protective compound from pest and disease, known as bioactive compounds. In this study, the quantitative phytochemical analysis was purposely conducted to determine the level of antioxidants and content of flavonoid and phenolic compounds, specifically gallic acid and quercetin. These analyses were performed using the DPPH method, and concentration of gallic acid and quercetin were analyzed using HPLC.\n\nBased on the results of total antioxidant values of wild mangoes using DPPH assay, it was revealed that Mangifera sp1. (MBS) is the highest antioxidant activity amongst other wild mangos (bark, 0.88 ppm; leaves, 33.24 ppm). According to Badarinath31, antioxidants are expected to be very strong if the IC50 value is smaller than 50, in the range of 50–100 ppm, yet it is weak when the IC50 value ranges between 100–250 ppm and is inactive if the IC50 is <250 ppm. The smaller the IC50 value, the higher the antioxidant activity. The antioxidants contained in mangos are essential for enhancing the immune system in the body, and are an immunomodulator agent32, including analgesic33, antimicrobial and antidiabetic properties34. Fitmawati et al.35 reported that powder ethanol extract of Mangifera sp1 (MBS) leaves are immunomodulatory agents with phagocytic activity at 69.67%. Ascorbic acid contained in the bark and leaves of mangos has great potential to heal chronic wounds and inflammation36. Also, mangos are a particularly rich source of polyphenols, a diverse group of organic micronutrients found in plants, which exert specific health benefits.\n\nThe presence of gallic acid and quercetin from wild mangoes in this study shows that they have potential as antioxidants of phenolic and flavonoid groups. The highest phenolic compounds of wild mangos were in the bark of M. foetida (var. batu) and leaves of M. torquenda (100.65 mg/g and 92.48 mg/g, respectively). Whereas, the lowest phenolic compounds of wild mangos were in the bark of M. foetida (var. limus) and the leaves of M. kemanga (41.76 mg/g and 20.08 mg/g).\n\nIn a previous study on the bark, old leaves, and young leaves of Van Dyke cultivated mango in Brazil, gallic acid content was 0.24 g/kg, 0.43 g/kg, and 3.49 g/kg, respectively11. The results of the present study show that wild mangos have higher phenolic contents than cultivated mangoes, such as the Van Dyke mangos. With the abundant phenolic content, wild mangos from Sumatra have a potential source of natural antioxidant agents. Currently, herbal tea derived from mango leaves (M. indica) has been developed and scientifically proved to be a source of mangiferin and other phenolic contents37,38. Therefore, phenolic contents found in wild mangos in this study may be expected to be further developed into healthcare products in the future.\n\nThe highest amount of flavonoid compounds were found in the bark and leaves of M. sumatrana (98.69 mg/g and 107.50 mg/g, respectively), and the lowest values were found in the bark of M. laurina and leaves of M. kemanga (63.9 mg/g and 48.55 mg/g, respectively). Barreto et al.11 reported that quercetin pentoside compounds in methanol extract of leaves of Van Dyke cultivated mangos from Brazil was 1.33 g/kg in old leaves and 4.93 g/kg in young leaves. In contrast, pentoside was undetectable in the bark11. However, the present study revealed that all bark of wild mangos assessed contained quercetin. Ali et al.39 and Kim et al.14 also reported that cultivated mangoes (M. indica) mostly contain flavonoids, carotenoids, vitamin E and C, terpenoids and steroids.\n\nBased on the results of the HPLC chromatogram, the contents of the bioactive compounds of wild mangos showed that the content of gallic acid obtained from wild mangoes leaves ranged from 5.22–35.47 mg/g of dry weight, where the lowest content was in M. sumatrana and the highest was M. foetida (var. manis). According to Soong and Barlow40, mango seed extract (Mangifera indica L.) contains gallic acid (23–838 mg/100 g of dry weight, equivalent to 0.23–8.38 mg/g). Rastraelli et al.41 also stated that gallic acid content in mango bark is ~226.2 mg/100g of dry weight, 6.0 mg/100 g in seed kernel42, and 6.9 mg/kg in mango pulp43. These results indicate that gallic acid content is higher in wild mango leaves (as seen in the present study) compared to other parts of the mango.\n\nThe study by Barreto et al.11 reported that gallic acid is the major secondary metabolite component in the flesh and seeds of mangoes (M. indica). Gallic acid is the most abundant molecule in ‘Ataulfo’ mango peel44. The flesh, seeds, and kernel of M. indica are sources of gallic acid, which is a bioactive compound with potential health-promoting activity45. Ajila and Prasada46 stated that quercetin is the major flavonoid type in the ripe peel of mango.\n\nGallic acid has anticancer, anti-inflammatory, antimicrobial, and antimutagenic, including radical-scavenging, activities47. It has also been shown to inhibit colon cancer cell (HCT-15) proliferation, inhibits platelet aggregation, calcium mobilization, and tyrosine protein phosphorylation in platelets16, and inhibits inflammatory allergic reactions48.\n\nQuercetin plays a role in triggering fruit color development. In the present study, M. laurina had the highest quercetin content, while the lowest was found in M. foetida (var. Limus). Quercetin content of wild mangoes leaves ranged from 0.76 to 1.47 mg/g of dry weight. Berardini et al.7 reported that quercetin content in mango peel was 65.3 mg/kg of dry matter, equivalent to 0.653 mg/g dry weight. Based on these data, wild mangos leaves have higher quercetin content (as found in the present study) than peel. High quercetin content is found in fruits and quercetin-3-glucoside is mainly found in leaves16.\n\nQuercetin has various medicinal benefits, including aiding treatment in brain disorders, renal injury, cardiovascular diseases, high blood pressure, cancer, bacterial activity, inflammation, diabetes mellitus, arthritis and asthma49–51. It decreases estrogen receptor cells of breast cancer, inhibits tyrosine kinase, arrests human leukemic T cell development52, protects the liver from oxidative damages53, prevents cardiovascular disease, and exhibits antihistamine and anti-inflammatory effects associated with various forms of arthritis16.\n\nFitmawati et al.35 demonstrated that wild mangos leaves from Sumatra have immunostimulant activity and antioxidant compounds54, including therapeutic efficacy potential for the prevention of degenerative diseases. Wild mangos from Sumatra contain flavonoids in high amounts, hence these contents need to be further explored and maximized for medicinal usage, in order to support the availability of medicinal resources. The results of the antioxidant activity using the DPPH method as assessed in the present study, and the total content of flavonoids and phenolics exhibited the varied results for each species. Wild mango species that have the highest antioxidant content may not have the highest total levels of flavonoids and phenolics because each plant species has different levels and types of metabolites.\n\n\nConclusions\n\nThis study provided quantitative information about the presence of antioxidants, as well as the specific antioxidant contents, of wild mangos. The current investigation was undertaken to quantify the percentage of phytochemicals in the leaves and bark of wild mangos as an alternative raw material for medicine. This showed that this study provides new knowledge regarding the content of gallic acid and quercetin in wild mangos, found in the leaves, which could be used as an immunomodulatory agent. The results obtained are expected to be useful in supporting the development of anti-degenerative drugs, as well as to support the conservation of wild mangos, which are rare while maintaining and improving their quality and diversity.\n\n\nData availability\n\nOpen Science Framework: Wild Mango Exploration in Sumatra for Potential Health Medicine, https://doi.org/10.17605/OSF.IO/MP6KF55.\n\nThis project contains the following underlying data:\n\n- Raw_Data_for_Gallic_Acid\n\n- Raw_Data_for_Quercetin\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "References\n\nFitmawati, Harahap SP, Sofiyanti N: Phylogenetic analysis of mango (Mangifera) in Northern Sumatra based on gene sequences of cpDNA trnL-F intergenic spacer. 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Reference Source\n\nSyahputra RF, Resida EF, Kholifah SN, et al.: Wild Mango Exploration in Sumatra for Potential Health Medicine. 2020. http://www.doi.org/10.17605/OSF.IO/MP6KF" }	[ { "id": "61846", "date": "04 May 2020", "name": "Sarifah Nurjanah", "expertise": [ "Reviewer Expertise Active compound of agriculture product" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe in detail a new information on antioxidant profile of 11 Sumatran wild mangoes (Mangifera spp), including flavonoid and phenolic content as well as gallic acid and quercetin of its leaves and barks. The paper is well written. A few suggestions are made, however:\nIntroduction: Need information why antioxidant activity is determined on leaves and barks instead of mango’s peel, pulp and seed kernel.\nMethod :\nThere is no information about quercetin and gallic acid standard for HPLC analysis. Need to specify the origin of quercetin and gallic acid standards.\n\nNo information of HPLC column, please specify the column used.\nResults and discussion :\nThe last sentence of paragraph 6 stated “Ailla and Prasada46 stated that quercetin is the major flavonoid type in the peel of mango”, since this paragraph talk about gallic acid not quercetin, so discussion about quercetin should be moved in the paragraph 8.\n\nOn paragraph 8 stated the lowest quercetin was found in M foetida (var Limus), actually as we can see from Table 3, it should be M foetida2 (var. manis) and M foetida3 (var. batu).\n\nTotal phenolic is represented by gallic acid content. Increasing phenolic content should increase the gallic acid, but the study didn’t show this. Why this condition can appear? Does any others phenolic compound instead of gallic acid influence the total phenolic in mango barks and leaves?\n\nFlavonoid content is represented by quercetin. Increasing flavonoid content should increase the quercetin, but the study didn’t show this. Why this condition can appear? Does any others flavonoid compound instead of quercetin influence the flavonoid compound in mango barks and leaves?\n\nIC50 values indicate the antioxidant activity, the lower IC50 value the higher antioxidant activity. In this study the antioxidant activity is represented by total phenolic and flavonoid content. Lowering IC50 should increase total phenolic and flavonoid content, but the study didn’t show this. Please discuss this phenomena.\n\nIs the work 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": "5501", "date": "20 May 2020", "name": "Fitmawati Fitmawati", "role": "Author Response", "response": "We would to thank the reviewer for their comments to our manuscript. In this new version, we have reviewed and modified text to improve the clarity and understanding manuscript. The main changes have been:1). Introduction the reason for using leaves and bark to determine the antioxidant value of wild mangoes is added.2). The method has already mentioned the origin of the standard quercetin and gallic acid and the HPLC column used.3). In the results and discussion of data changes due to input errors in the previous article. It has been discussed that there are other compounds of flavonoid and phenolic derivatives in leaves and bark of wild mango that refer to the literature, thus affecting the amount of gallic acid and quercetin, as well as antioxidant values." } ] } ]	1	https://f1000research.com/articles/9-220
https://f1000research.com/articles/9-661/v1	30 Jun 20	{ "type": "Case Report", "title": "Case Report: Sarcoidosis with azygos vein enlargement mimicking metastatic cancer", "authors": [ "Abdallah Qasim", "Omar Kousa", "Mohamed Mansour", "Ahmad K. Aly", "Dana Awad", "Hamza Kousa", "Yazan Addasi", "Bader Abuhazeem", "Venkata Andukuri", "Omar Kousa", "Mohamed Mansour", "Ahmad K. Aly", "Dana Awad", "Hamza Kousa", "Yazan Addasi", "Bader Abuhazeem", "Venkata Andukuri" ], "abstract": "Sarcoidosis is a systemic disease with heterogeneous clinical manifestations that is characterized histologically by the presence of noncaseating granulomas in the affected organs. It can be a diagnostic challenge, especially when mimicking malignancy or fungal infections. Previous case reports of sarcoidosis presenting with multiple masses are highly suggestive of infectious or malignant etiology. In this case, our patient presented with enlarged lymph node and was found to have innumerable nodules in the mediastinum, lungs, and liver. Azygos vein enlargement was also seen on radiological imaging, and malignancy was highly suspected; hence, an extensive workup was conducted, including laboratory, radiology and biopsy evaluation, which were diagnostic of sarcoidosis. Our case showed the importance of correlation of the history, physical examination, radiological and histopathologic studies in confirming the diagnosis and the need to rule out other serious infections and malignancies, especially with azygous vein enlargement, which can sometimes be missed in chest radiograph.", "keywords": [ "Sarcoidosis", "Malignancy", "Liver mass", "Azygous vein" ], "content": "Introduction\n\nSarcoidosis is systemic inflammatory condition of unknown etiology that is characterized histologically by the presence of noncaseating granulomas in the affected organs. It is called the ‘great mimicker’ because it can present in various ways similar to other diseases. It can affect multiple organ systems, but most commonly involves the lungs and the lymph nodes. Other less commonly involved organs include the eyes, skin, liver, spleen, bone marrow, heart and brain1,2. The age adjusted annual incidence of sarcoidosis in the united states is 35.5 per 100,000 for African Americans compared with 10.9 per 100,000 for Caucasians, according to a five-year cohort study3. We present a case presented with asymptomatic cervical lymph node enlargement, multiple liver lesions and azygos vein enlargement, which were suggestive of malignant or infectious etiologies. However, investigations resulted in the diagnosis of the “great mimicker disease’ sarcoidosis.\n\n\nCase presentation\n\nA previously healthy 53-year-old Caucasian man on no regular medication presented to his primary care provider complaining of a painless slowly growing cervical lump that he noticed one month prior to presentation. Review of systems were negative for fever, weight loss, night sweat, fatigue, loss of appetite, cough, chest pain, hemoptysis, or shortness of breath. Family history was significant for lymphoma. The patient never smoked, and was not exposed to any chemicals at work. Physical examination showed normal vital signs and enlarged right cervical lymph nodes. The rest of the physical examination were unremarkable. Initial laboratory workup showed normal complete blood cell count and basic metabolic panel. (Table 1). A decision to proceed with fine needle aspiration was taken, which showed a non-necrotizing inflammatory reaction. Therefore, a core biopsy was recommended to rule out lymphoma and he was referred to the rheumatology clinic for possible sarcoidosis.\n\nThree months after the initial presentation, he was seen in the rheumatology clinic; further workup was ordered, including an angiotensin-converting enzyme level which was normal, and chest radiograph (CXR), which showed azygous vein enlargement and thus a high suspicion of malignancy (Figure 1). Next, computed tomography (CT) scan of the chest was done and it showed pathologic lymph node enlargement within the thoracic inlet and mediastinum, innumerable bilateral small pulmonary nodules, and multiple liver cystic lesions concerning for metastatic disease. (Figure 2–Figure 4). Further metastatic workup with CT abdomen/pelvis showed multiple hepatic cysts without any other organ involvement (Figure 5). At the same time, histoplasma antigen/antibody were negative, and his erythrocyte sedimentation rate was within normal limit. (Table 2)\n\nPossibly Azygous vein enlargement.\n\nSagittal (A) and coronal (B) images in lung window showing multiple nodules in centrilobular (white arrowheads) and subpleural (black arrowheads) location consistent with peri lymphatic distribution that can be seen in cases of sarcoidosis.\n\nIncidental liver cysts are also noted.\n\nOne month later, the patient was referred to the pulmonary clinic and underwent general surgery for an excisional biopsy on the most accessible lymph node, which showed non-caseating granulomas without evidence of malignancy, fungal or mycobacterial infection, and confirmed the diagnosis of sarcoidosis. The patient was subsequently started on tapered prednisone (40 mg for two weeks then 20 mg for two weeks then 10 mg for one month). Three months later, he had repeated imaging which showed the same lesions in the lungs and the liver that are of the same size. Therefore, as he was completely asymptomatic, a decision to discontinue prednisone with 1-year radiology imaging follow-up was taken.\n\n\nDiscussion\n\nThe clinical manifestations of sarcoidosis vary and can range from asymptomatic disease detected incidentally on imaging studies to the presence of constitutional symptoms and symptoms attributed to the organ system involved, to multiorgan failure1,2. The diagnosis of sarcoidosis requires fulfillment of certain criteria, which include existence of typical clinical and radiological findings, histopathological evidence of noncaseating granulomas, and exclusion of other causes of granulomatous inflammation4.\n\nThe azygos vein is formed by the union of the ascending lumbar vein and the right subcostal vein. It is usually too small to be seen on chest x-ray. The enlargement of the azygous vein on chest x-ray can be seen in multiple diseases, such as congestive heart failure, inferior vena cava thrombosis, and intrathoracic malignancy5.\n\nIn this report, we present a patient with asymptomatic cervical lymph node and azygos vein enlargements, with innumerable lung bilateral nodules, hilar, para-esophageal and mediastinal lymph node enlargement and multiple liver nodules which were highly suspicious for malignancy or fungal infection. However, histological evaluation was consistent with sarcoidosis. To our knowledge, this is the first reported case of sarcoidosis and azygous vein enlargement. Hence, we conducted a systematic review of the literature for studies published from 1960 to October 2019 in PubMed, Scopus, Web of Science, and Cochrane Central databases. The following search terms were used: “sarcoidosis”, “azygous vein enlargement’ and “malignancy”. Our search was limited to individuals aged 18 years and older. Our search revealed a total of three patients. None of the cases showed azygous vein enlargement.\n\nOur findings were similar to previous cases reported by Oketani et al.6 who were among the first to report a case of sarcoidosis mimicking metastatic cancer. They reported a case of a 49-year-old female who was found to have mediastinal and intraabdominal lymphadenopathy on radiologic imaging, in addition to presence of space occupying lesions in the liver and the spleen, which raised suspicion for metastatic hepatocellular cancer. However, histologic examination of liver biopsy specimen showed evidence of noncaseating epithelioid granulomas suggestive of sarcoidosis, which responded to treatment with steroids. Giovinale et al.7 reported another case of a 55-year-old female who presented with weight loss and abnormal liver function tests. Imaging revealed the presence of abdominal lymphadenopathy, as well as hepatic and splenic lesions, which were thought to be related to metastatic cancer, as the patient has family history of colorectal cancer. However, histological examination of specimens obtained during exploratory laparotomy showed chronic granulomatous inflammation, and the work up for neoplasia was negative7. Finally, Jafari et al.8 reported a case of a 39-year-old male who was found to have hilar lymphadenopathy and a hepatic nodule on radiologic imaging, which were initially thought to be related to metastatic cancer; however, biopsy confirmed chronic granulomatous inflammation and a diagnosis of sarcoidosis was made after ruling out tuberculosis.\n\n\nConclusion\n\nAzygous vein enlargement is a commonly missed finding on chest radiograph which can be a sign of underling malignancy. However, it can also be seen in sarcoidosis. Further appropriate tests are required to confirm the underlying etiology.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patient/parent/guardian/relative of the patient.", "appendix": "References\n\nNewman LS, Rose CS, Maier LA: Sarcoidosis. N Engl J Med. 1997; 336(17): 1224–1234. PubMed Abstract \| Publisher Full Text\n\nThomas KW, Hunninghake GW: Sarcoidosis. JAMA. 2003; 289(24): 3300–3. PubMed Abstract \| Publisher Full Text\n\nRybicki BA, Major M, Popovich J, et al.: Racial Differences in Sarcoidosis Incidence: A 5-Year Study in a Health Maintenance Organization. Am J Epidemiol. 1997; 145(3): 234–241. PubMed Abstract \| Publisher Full Text\n\nStatement on Sarcoidosis: American Journal of Respiratory and Critical Care Medicine. 1999; 160(2): 736–755. Publisher Full Text\n\nShin M, Ho K: Clinical significance of azygos vein enlargement. Clin Imaging. 1999; 23(4): 236–241. Publisher Full Text\n\nOketani M, Tsubouchi H, Hori T, et al.: Sarcoidosis with tumorous hepatic and bone lesions mimicking disseminated malignancy: A case report. Gastroenterol Jpn. 1992; 27(3): 414–417. PubMed Abstract \| Publisher Full Text\n\nGiovinale M, et al.: Atypical sarcoidosis: case reports and review of the literature. Eur Rev Med Pharmacol Sci. 2009; 13 Suppl 1: 37–44. PubMed Abstract\n\nJafari B, Sabz G, Masnavi E, et al.: Case Report: Pulmonary and Liver Sarcoidosis Suspected of Metastasis. F1000Res. 2018; 7: 288. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "71327", "date": "18 Sep 2020", "name": "Marios Rossides", "expertise": [ "Reviewer Expertise Medicine and Clinical Epidemiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this case report, the authors describe a case of a man in his 50s with sarcoidosis presenting with a cervical lump (enlarged lymph node), multiple abnormalities in CT chest and abdomen suspected for malignancy and azygos vein enlargement in chest X-ray.\n\nThe case report is complete with all necessary information on clinical presentation, history, examination, and necessary imaging and lab results and follow-up of up to a year. Reporting of imaging and lab findings is of adequate quality. The authors also conducted a literature search to identify similar cases.\n\nAlthough it is common practice to exclude malignancy during sarcoidosis diagnosis, this report highlights the fact that azygos vein enlargement may mislead physicians and increase the suspicion for malignancy and away from sarcoid granulomatous inflammation. Raising awareness on this issue is clinically useful.\n\nThere are a few minor issues with this case report that the authors should address:\nIntroduction: Are there any more contemporary estimates of sarcoidosis incidence? The article the authors cited is from 1997.\n\nWere pulmonary function tests performed in this patient?\n\nCould the authors explain why the patient was started on prednisone?\n\nDiscussion: The authors mentioned that “the diagnosis of sarcoidosis requires fulfillment of certain criteria […]”. There are no evidence-based ‘criteria’ for diagnosing sarcoidosis and authors should rephrase to make this explicit.\n\nDiscussion: Azygos vein is misspelled as “azygous” vein; it should be corrected.\n\nConclusion: The authors stated that azygos vein enlargement is usually missed in chest X-rays but do not provide any reference to support this statement.\n\nFigure 1: All abnormalities seen on the chest X-ray should be described in the figure legend.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "78482", "date": "08 Feb 2021", "name": "Ichiro Mizushima", "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\nIn this report, the authors described a male patient histologically diagnosed with sarcoidosis, which might have caused azygos vein enlargement. He was treated with glucocorticoids, but his lung and liver lesions did not respond to the treatment. Although this is an interesting case, the following concerns in addition to the comments of Reviewer 1 should be addressed for better understanding of this case:\nIn this case, the liver lesions are described as ‘cystic lesions’ or ‘cysts’, which generally contain air, fluids, or semi-solid material, but not massive cells. In addition, the liver lesions did not respond to glucocorticoids. Therefore, the authors are recommended to explain whether the authors diagnosed the liver lesions as sarcoidosis-associated lesions or not, and if they did, the criteria based on which the liver lesions could be diagnosed as sarcoidosis-associated.\n\nIn this case, the lung lesions had multiple nodules, which could be consistent with sarcoidosis-associated lung lesions. However, the lung lesions did not respond to glucocorticoids. Therefore, it is necessary for the authors to clarify whether the authors diagnosed the lung lesions as sarcoidosis-associated lesions or not.\n\nLymphadenopathy was histologically confirmed to be associated with sarcoidosis in this case. Azygos vein enlargement was presumed to be caused by mediastinal lymphadenopathy. However, the response of the lymph node lesions and azygos vein enlargement to treatment with glucocorticoids was not described in the original manuscript. The authors should clarify these issues.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "6642", "date": "05 May 2021", "name": "Abdallah Qasim", "role": "Author Response", "response": "Thank you for your time and efforts to evaluate this case report. 1 - The cystic appearance of the lesions in the liver is what made malignancy on top of our differential diagnosis which prompted us to do an excisional biopsy of one of the accessible lymph nodes that did not show malignancy and showed evidence of non-caseating granulomatous disease. Work up for other granulomatous diseases was negative. the diagnosis was made that this is a case of sarcoidosis. And looking in the literature there were cases reported of atypical hepatic sarcoidosis lesions that were cystic; Giovinale M, Fonnesu C, Soriano A, Cerquaglia C, Curigliano V, Verrecchia E, De Socio G, Gasbarrini G, Manna R. Atypical sarcoidosis: case reports and review of the literature. Eur Rev Med Pharmacol Sci. 2009 Mar;13 Suppl 1:37-44. PMID: 19530510. 2 - Even though the lesions didn't respond to steroid, we still think that he has steroid-resistant sarcoidosis and the lesions in the lung are due to sarcoidosis and most likely the liver lesions as well. and because the patient is asymptomatic so the decision was made to observe the patient. 3 - The azygous vein enlargement didn't change after treatment with steroids as the lung and liver lesions." } ] } ]	1	https://f1000research.com/articles/9-661

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